POLITECNICO DI MILANO

DIPARTIMENTO DI FISICA

PhD course in Physics

XXV cycle

DEVELOPMENT AND APPLICATION OF A RAMAN

MAPPING INSTRUMENT FOR THE STUDY OF

CULTURAL HERITAGE

Supervisor: Prof. Gianluca Valentini

Tutor: Prof. Rinaldo Cubeddu

PhD coordinator: Prof. Paola Taroni

Candidate: BRAMBILLA ALEX

2010 - 2012

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“In all things of nature there is something of the marvelous”

Aristotle

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Summary.

Summary. 4

Abstract. 7

Introduction. 10

CHAPTER I

RAMAN SPECTROSCOPY. 13

I.1 Molecular vibrations. 13

Vibrational energy. 14

Normal modes of vibration. 15

I.2 Classical theory of Raman scattering. 19

Susceptibility and polarizability. 20

Rayleigh and Raman scattering. 21

I.3 Quantum theory of Raman scattering. 24

Time dependent perturbations: the second order transition probability. 25

A semi-classical expression for Raman effect. 28

Full quantum approach. 32

Second quantization. 33

The Kramers-Heisenberg formula. 36

General considerations. 39

I.4 The cross section. 41

I.5 Surface Enhanced Raman Scattering. 43

Bibliography. 45

CHAPTER II

THE REMOTE RAMAN SCANNER. 47

II.1. Raman spectroscopy in Cultural Heritage. 47

II.2. The aim of the project. 49

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Raman imaging. 50

Remote Raman sensing. 51

Portable Raman instruments. 53

II.4. Remote Raman Scanner: the layout. 55

II.5. RRS: the standard components 57

Semiconductor laser. 57

Galvanometric mirrors. 59

Holographic notch filter. 60

Czerny-Turner spectrograph. 60

Charge coupled device (CCD). 61

Optical fibers. 62

II.6 RRS: the custom-made systems. 63

The optical system. 63

The software. 67

Bibliography. 71

CHAPTER III.

RRS AT WORK: TESTS AND CASE STUDIES. 77

III.1 Characterization of the performances. 77

Assessment of the depth of the field of view. 78

Assessment of the field of view. 79

Assessment of the spectral accuracy. 80

Assessment of the spatial resolution. 80

III.2 A multi-analytical approach to a model painting. 82

Reflectance spectra and reconstructed maps. 83

XRF analysis. 87

Flexible Raman mapping. 91

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III.3 Identification of white and fluorescent pigments. 94

III.4 Analysis of samples from the “Memoriale italiano” of Auschwitz. 97

III.5 Study of a 3D plastic object 100

III.6. Mapping of a rock sample. 102

Bibliography. 106

CHAPTER IV

SERS ANALYSIS OF AMINO ACIDS WITH A PORTABLE INSTRUMENT.

108

IV.1. The aim of the work. 108

IV.2 SERS of amino acids. 109

IV.3 SERS with metal nanoparticles. 112

IV.4 The portable instrument. 115

IV.5 Tests and results. 116

Raman spectra of amino acids. 117

Analysis of liquid samples. 120

Analysis of dried solutions. 122

Dependence of L-Met spectra on pH. 124

Distinguishing two amino acids. 125

Towards a more complex sample. 128

IV.5 Perspectives. 129

Bibliography. 135

CHAPTER V

CONCLUSION. 140

Acknowledgements. 144

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Abstract.

Raman spectroscopy is a consolidated technique for the analysis of materials: it is

based on the identification of the frequency shifts undergone by light scattered by a

molecule; this behavior is due to an interaction of the incident photons with the

vibrational quanta of the involved material. The possibility to obtain information on

a molecular scale about the chemical identity of a sample in a non-destructive way,

makes this method one of the favourite for the analysis of valuable objects, as it is in

case of Cultural Heritage. In this field of application it is particularly urgent the

necessity to monitor wide surfaces of heterogeneous objects often with an irregular

shape. Compared to a laboratory measurement, moreover, an in situ analysis implies

further requirements due to the dimensions and the portability of the employed

instrumentation, which must guarantee an adequate sensitivity to Raman signal

while keeping the necessarily limit of a non-invasive procedure.

To answer to these needs, a portable Raman spectroscopy system which works with

a non contact approach, has been conceived, designed and assembled. The device is

able to analyze a surface 5 cm wide placed at a distance of 20 cm with a flexible

mapping approach: a pair of galvanometric mirrors allows the deflection of the laser

beam, which is focused on the point of interest by a dedicated optical system. This

element was tailored to collect and guide the light scattered by the sample towards

the instruments of analysis (spectrograph and charged coupled device) which, like

the laser source, are remotely positioned and connected by optical fibers. This

solutions permits to keep the measuring probe light and small, and to mount it on a

tripod in order to ensure maximum flexibility and stability at the same time; the

instruments of analysis can be, alternatively, connected to a more conventional

Raman microscope when a deeper punctual analysis is needed. The chance of

performing remote measurements not only considerably widens the range of object

to study, but it also makes the device less affected by the vibrations which perturb

contact analysis when performed out of the laboratory environment; moreover, the

particular combination of the chosen optic elements ensures a depth of field ideal

for the analysis of three-dimensional objects.

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The thesis treats the theoretical foundations of Raman spectroscopy to highlight its

potentialities and stress the common problematiques (in primis the low efficiency

with respect to competitive phenomena such as fluorescence); then, the phases of

the project of the Remote Raman device are reported together with the methods to

test its performances and features. One chapter is dedicated to the results of

punctual analysis and to the study of wide areas in samples of artistic or cultural

interest; whenever it is possible, these are compared to the information obtained by

other non-invasive techniques on the same objects. Last, surface-enhanced Raman

measurements on amino acids in low concentrations are reported as a preliminary

study for the analysis of organic traces in archaeological remains.

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Introduction.

The application of scientific techniques to the study of Cultural heritage is wide

reaching. Not only research groups of chemists, physicists, geologists, engineers and

biologists are growingly demanded to provide diagnostic methods for several kinds

of artefacts, but museums and conservation centers now rely on scientific pools of

specialists equipped with cutting-edge analytical tools. The information furnished by

analytical methods, yielding data on the atomic and the molecular composition

through spectroscopic techniques, for example, is valuable not only for historical or

scientific interest, but also important as data can support critical evaluation, the

authentication of a masterpiece, or guide the restoration operation.

Despite the fact that the role of scientific methods in humanistic studies such as

preservation, conservation and promotion of cultural heritage is now undisputed,

the research of new methods of application continues to develop. The typical issues

related to the analysis of cultural heritage, which generally concern the need to

interact in the softest possible way with the object of interest, represent a

continuous challenge for researchers, who look for techniques capable of providing

significant information with a non-destructive or, preferably, a non-invasive

approach. These two requirements have lead to the spread of optical analytical

techniques, like reflectance spectroscopy, Infrared absorption spectroscopy and

Raman spectroscopy, for the characterization of surfaces of works of art; in most of

their applications, these methods rely on a laser as a light source, therefore they

benefit from the explosion in development and expansion of these instruments in

the last decades. The parallel improvement and the declining price of light detectors,

in addition, has led to the birth of imaging versions for optical analyses: the

possibility of studying a large set of points on a surface with a single acquisition

further promotes the application of these techniques to the study of artworks.

Within this framework, this thesis presents a novel instrument for Raman

spectroscopy, able to perform a stand-off mapping of a macroscopic surface at a safe

working distance of approximately 20 cm and thus particularly suitable for

analyzing valuable objects. Commercially available Raman Spectrometers which are

marketed for the analysis of works of art work either in contact or at very short

working distances (on the order of millimeters). The aim of the reported work has

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been to build an instrumental apparatus ready to be operated in the laboratory as

well as on site; the need for a significant flexibility in the measurement of different

points of a representative area, led to the design and development of a dynamic

mapping device, which can selectively illuminate and stimulate the Raman

scattering of each point within its field of view, without any relative movement

between the probe head and the target object. The layout of the set-up has been

oriented towards a remote instrument, capable of being employed even in

uncomfortable environments thanks to a portable probe head and to a working

distance of 20 cm, sufficient to ensure a non-invasive approach to the object of

interest, and at the same time to reduce the sensitivity to vibrations.

A thorough study of Raman scattering has been the first, indispensible step in the

design of the measurement apparatus. In Chapter 1, a description of the Raman

effect, as explained by the classic and the quantum theory, is presented, based on

some of the most common approaches followed in physics and chemistry. Particular

mention to the standard Raman event and to the enhancement to the effect provided

by the interaction with a metal substrate is made; one of the aims of the thesis,

indeed, is to determine how the adoption of suitable procedures and the

optimization of technological parameters can help the detection of the intrinsic

weak Raman scattering with a portable apparatus, whose acquisition is generally

more critical of bench-top instrument. In Chapter 2 the state of the art of Raman

mobile and stand-off devices is addressed; this study compares the proposed set-up

with available Raman mapping and imaging devices. In addition the choices

determined during the design and construction of the system are reviewed. With

Chapter 3, the performances of the completed set-up are reported: in this case, the

focus is on the capability of providing the identification of different kinds of

materials and samples of heterogeneous objects. The integration of the Raman

mapping instrument with other non-destructive analytical methods is evaluated on

a model of a painted panel, as the ultimate test of the application. In Chapter 4 tests

performed with a commercial mobile Raman instrument, chosen for a different kind

of analysis are reported: the study of organic fragments contained in ancient vessels

and pottery. For this specific purpose, research was carried out with a device with a

portable probe head but based on the traditional microscope layout. The novelty of

the chosen approach resides in the adoption of metal nanoparticles as a substrate to

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enhance the Raman cross section of the analytes, in this case simulated by solutions

of amino acids. The exploited technique, which goes under the name of Surface-

Enhanced Raman Spectroscopy (SERS), has been applied for the first time with a

portable Raman instrument on this kind of materials: amino acids and a small

peptide, in small concentrations have been chosen for the preliminary tests to assess

the detection limits for these organic materials. The reported results are the first

step towards the determination of a routine method that could be easily applied in

situ to reveal traces of biological materials on archaeological finds.

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CHAPTER I

RAMAN SPECTROSCOPY.

Raman spectroscopy is based on the homonym effect, postulated by Smekal in 1923

[I.1] and first observed experimentally in 1928 by C.V. Raman and Krishnan [I.2]: in

the original experiment, for which Raman was awarded the Nobel prize in physics

two years later, sunlight was focused by a telescope onto a sample which was either

a purified liquid or a dust-free vapor. A second lens was placed by the sample to

collect the scattered radiation and a system of optical filters was used to show the

existence of scattered radiation with an altered frequency from the incident light –

the basic characteristic of Raman spectroscopy [I.3]. Inelastic scattering is due to the

interaction of the electromagnetic radiation with the level structure of a molecule or

a solid: the scattering event corresponds to a virtual transition (in a sense that will

be specified later) between rotational, vibrational or electronic levels triggered by

light and mediated by quanta of electromagnetic energy called photons. Commonly,

Raman spectroscopy is focused on the second type of these processes, involving

vibrational transitions. Together with infrared absorption, this technique is used to

obtain information about the structure and properties of molecules and solids in a

non destructive way [I.4].

I.1 Molecular vibrations.

A molecule is classically described as the collection of M nuclei and N electrons [I.5]

arranged in a stable configuration: if the positions of the nuclei are considered fixed,

each molecule can be associated to a “molecular point group”, which is a symmetry

group corresponding to a fixed point, the center of mass of the molecule. In the same

way, a crystal is described as the periodic distribution of a unit cell, made out of a set

of atoms, along each point of a spatial lattice, called Bravais lattice, generated by

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three linearly independent vectors. Each crystal is associated to a crystallographic

group according to the symmetry of the point group of its cell, and to the symmetry

of the lattice [I.6]. The symmetry properties of molecules and solids are determinant

for the vibrational transitions which are observed with Raman, and in general

vibrational spectroscopy; this is true also in a negative sense, as it is for amorphous

materials, in which regularities are mostly absent and a statistical description of the

structure is necessary.

For the present discussion, the molecule is considered as the model for the classical

and quantum systems on which the Raman effect is treated: this allows the

treatment to be relatively compact and still valid for gaseous, liquid and many solid

samples; whenever it is needed, reference to crystals and amorphous materials will

be explicitly made.

Vibrational energy.

A common approximation when describing the molecule is to consider that the

motion of the electrons can be separated by the motion of nuclei. This assumption

(called Born-Oppenheimer approximation) is realistic, considering the ratio of the

masses of the electron and a generic nucleus: the motion of the electrons is faster

than the one of nuclei at least by 3 orders of magnitude, and so the latter are almost

“at rest” with respect to these particles. Thanks to Born-Oppenheimer

approximation, the problem to determine the dynamic of the electrons is solved

while maintaining fixed the positions of the nuclei (which appear in the solution as

parameters) and the electronic contribution obtained by this calculation is used to

describe the total energy of the system [I.5]. This separation of the electronic motion

and the nuclear motions is only an approximation which may break down in certain

cases, especially for high electronic states. If there were no interaction between the

two types of motions, there would be no Raman effect of any importance. However,

the coupling is small for the lowest electronic state [I.7].

The molecule, seen as a rigid body, can translate along three arbitrary Cartesian

axes: therefore, three of its 3M degrees of freedom represent its translations in

space. If the center of mass is then fixed, the molecule has 3M – 3 remaining degrees

of freedom: rotations around the coordinate axes represent three (or two, for a

linear molecule) of these degrees of freedom. Six (five) parameters are needed, thus,

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to describe the molecule as a whole: the remaining 3M – 6 (or 3M – 5) degrees of

freedom describe, instead, the variations among the “standard” location of the nuclei

around the center of mass, i.e. the deformations of the molecule: these are called

vibrational degrees of freedom. The global energy of the isolated molecule can be

accordingly divided into four terms:

Emol = Eelectronic + Etranslational + Erotational + Evibrational (1.1)

From a quantum-mechanical point of view, each of this term is associated to a

component of the total molecular Hamiltonian.

ℋmol = ℋelectronic + ℋtranslational + ℋrotational + ℋvibrational (1.2)

Within the chosen approximation, the four terms are considered independent and so

the separation of variables within the Schrödinger’s equation for the molecule is

carried on, leading to a factorial expression for the wave function

Ψmol = Ψelectronic Ψtranslational Ψrotational Ψvibrational (1.3)

The reported considerations refer to a molecule in absence of an external potential:

further considerations are necessary to take into account the interaction of this

system with light, as it will become clearer in the following sections.

Normal modes of vibration.

The translational motion of the molecule does not ordinarily give rise to radiation.

Classically, this happens because acceleration of charges is required for radiation.

The rotational motion causes practically observable radiation if, and only if, the

molecule has an electric (dipole) moment. The vibrational motions of the atoms

within the molecule may also be associated with radiation if these motions alter the

electric moment [I.7].

Vibrations, as we have seen, correspond to any displacement among the positions of

the atoms which do not alter the position of the center of mass nor its angular

momentum. The dynamic description of these vibrations must consider, therefore,

forces internal to the molecule: at a first approximate analysis, the intra-molecular

potential can be limited to the nearest neighbor atoms. This is equivalent to consider

the intra-molecular potential V(R) of a biatomic molecule, which is, if referred to the

equilibrium distance R0 between the two atoms, clearly asymmetric (fig.I.1); the

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energy of the molecule tends to infinite if the atoms are too close, and instead

reaches a saturation for very long distances, when it is equal to the work necessary

to dissociate the molecule. Even though, in the range in which the molecule is stable

(below the dissociation value), it is possible to approximate the potential with a

harmonic oscillator. The expression of the intra-molecular potential can indeed be

expanded around its minimum, and it is convenient before performing this

operation, to introduce a suitable frame of reference.

Fig.I.1. The intra-molecular potential curve V(R) for a bi-atomic molecule (blue curve). Without any

loss of generality, in the graph the dissociation energy value is set equal to 0. The green curve refers

to the harmonic approximation.

To start with, a standard Cartesian system of coordinates xi is chosen. A molecule

constituted by M atoms is described by a set of 3M coordinates, so i runs from 1 to

3M; at the equilibrium configuration, these respectively assume the values ci. The

displacement along each coordinate, therefore, will be calculated as the difference

Δxi = xi – ci; clearly, the time derivatives (indicated by a dot over the letter) of the

initial Cartesian coordinates are exactly equal to the corresponding time derivative

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of the displacement coordinates. A set of the so-called mass-weighted coordinates qi

is constructed according to the relationship

q m

where, obviously, the mis are equal in groups of three in a row because they refer to

the same atom. This choice permits to simplify the kinetic energy expression

q

The series expansion of the potential energy is, accordingly, carried out in powers of

qi:

q

q

q q

q q

,

A comfortable choice of V0 is to set it to zero, without any loss of generality. The

second term in the right-hand side of the equation is automatically zero for the

definition of the equilibrium position (the derivative is evaluated for qi = 0). If the

expansion is arrested at the second order, therefore, only the last term is surviving,

and it can be synthetically written as

q q

q q

,

f q q

where the second order derivatives of V, evaluated in the equilibrium position, can

be considered as the elements fij of a 3M × 3M dynamic matrix F. The equations of

motion of the molecule are described by the solutions of the 3M Lagrange equations

d

dt

q

q

which, with the chosen approximation, can be written as a set of 3M simultaneous

second-order linear differential equations, each one closely resembling the well-

known equations of the harmonic oscillator:

q f q

(1.4)

(1.5)

(1.6)

(1.7)

(1.8)

(1.9)

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If a tentative solution of the form

q cos t

where Ai, and are properly chosen constants, is substituted, the set of differential

equations is transformed in a set of 3M simultaneous algebraic equations in Ai

f

Where the ij symbol stands for the Kronecker delta. The solutions of these

equations are not trivial only for a special set of s (which have the physical

dimensions of pulsations): clearly, this is but a secular equation for the dynamic

matrix F. For each particular value of , namely k, a certain set of amplitude Ai,

namely Aik, is obtained; however, it can be shown that only the ratios between the

different Ais is determined per each k. Therefore, the choice of convenience, if there

is no strict constraint given by the initial conditions on qi, is to set the values for

which the modulus of the vector [A1k, A2k, 3Mk] is unitary. These normalized

vectors are indicated as aik, and they can be grouped in a matrix called A.

It can be demonstrated, as well, that six out of the 3M eigenvalues of the matrix F are

zero: the null roots of the secular equation correspond to null pulsations, which

means that six degrees of freedom, as previously reported, are not ascribable as

vibrations. This means that, through a proper choice of the coordinates, this can be

reduced to a 3M – 6 square matrix without any loss of information. It can be easily

shown that this transformation is performed by multiplying the qi vector by the

matrix A of the normalized eigenvectors. Since there are only 3M – 6 eigenvectors,

each one made of 3M elements, the transformation produces naturally a new system

of 3M – 6 coordinates Qk, called vibrational coordinates. On the basis of the

vibrational coordinates, the dynamic matrix is diagonal, i.e. the harmonic oscillators

are decoupled.

This rather laborious analysis of the intra-molecular potential has the advantage to

introduce all the necessary elements to thoroughly describe molecular vibrations.

From the initial truncation of the series expansion, called Mechanic Harmonic

approximation, it was possible to derive the dynamic matrix F. The eigenvalues k of

this matrix correspond to the pulsations of the oscillations of the atoms around their

(1.10)

(1.11)

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equilibrium positions, their amplitude along the coordinate axis (except for a

multiplicative factor) given by the eigenvectors Aik. The equations (1.10) therefore,

describe motions in which all the atoms vibrate with a single frequency ωk = k½ in

phase: these are called vibrational modes of the molecule, and are described by the

vibrational coordinates Qk as independent harmonic oscillators.

Qk = Qk0 cos (ωkt + k) (1.12)

For the properties here described, the modes are orthogonal and normal: this

property allows the decomposition of any collective displacement of the nuclei of the

molecule in a series built over a basis of independent oscillations. In addition, it is

worth to anticipate that the decomposition along the normal modes of vibration is

necessary for the quantum description of Raman effect, because only within this

frame of reference the vibrational eigenfunction Ψvibrational can be written as the

product of independent quantum oscillators.

It is possible to calculate the force constants of each harmonic oscillator starting

from ab initio Hartree-Fock or density functional theory (DFT) methods: the

symmetry of the potential function will allow the reduction in size of the dynamic

matrix to a group of smaller matrices, one for each irreducible representation of the

molecular point group. Each normal mode of a molecule forms a basis for an

irreducible representation of the point group of the molecule itself [I.8]. Group

theory can be used to recover the normal mode coordinates starting from the

geometry of the molecule: the use of the so-called character table permits not only to

list the vibrational modes, but it also assigns them to the respective class of the

symmetry operations that build up the point group [I.8]. At the same time, the study

of the molecule symmetry allows the identification of degenerate vibration

frequencies and it is crucial for the determination of a mode’s activity in the Infrared

or Raman spectrum.

I.2 Classical theory of Raman scattering.

Raman effect is classically explained in terms of the dependence of the polarizability

of the molecule on the normal modes of vibration of the molecule. The scattering of

an electromagnetic wave, indeed, can be expressed in terms of the polarization

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induced in the material by the incoming electric field (the role of the magnetic field

can be neglected for the present treatment). It is known, in fact, that an oscillating

dipole absorbs end emits energy in the form of electromagnetic waves. Polarization,

indicated by the letter P, is a vector which describes the average (and hence,

macroscopic) contribution of the electric dipoles p which can be associated to the

molecules constituting the material. There are two possible models to describe this

quantity: one introduces the concept of susceptibility and is more suitable for

describing the behavior of a solid, being it an isolator or a metal; whereas for

molecules it is convenient to introduce the polarizability. Both these descriptions

consider only the linear dependence in the electric field E, but a more thorough

theory of scattering must include higher order terms1 as well (which are necessarily

taken into account for an explanation of phenomena such as hyper Raman or

stimulated Raman scattering, not considered here).

Susceptibility and polarizability.

The first approach follows a global description of the scattering material.

Polarization, then, can be written as the difference between the dielectric

displacement vector, D and the field in the vacuum multiplied by the permittivity of

the vacuum ε0. Polarization can be expressed, as well, making explicit the

proportionality to the electric field: the constant which relates the vector P and E is,

apart from a factor of ε0, known as susceptibility and written as χ

P = D - ε0E ε0 εR -1) E ε0 χ E (1.13)

χ is dependent only on the properties of the materials and on the frequency of the

electric field; in the most general case, where P is not necessarily parallel to E, it is a

tensor.

If the description of the electric induction is measured on the single molecule, it is

convenient to express the polarization in terms of the induced dipole momentum p;

1 The total time-dependent induced electric dipole moment vector of a molecule may be written as the sum of a series of time-dependent vectors as follows: p = p

(1) + p

(2) + p

(3) + …

where p(1)

= αE, p(2)

= ½ βEE, p(3)

= ⅙ γEEE …[I.12] and the order of magnitude of the addends

p(n) decreases dramatically with the order n.

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this is related to the electric field through the polarizability α, which is again a tensor

(p can be non parallel to E).

p = α E (1.14)

where α is measured in C m2/V unity and its components are indicated with the

notation αρσ, the indices ρ and σ assume the values x, y, z. Except in very special

circumstances, the Raman polarizability tensor is real and symmetric, i.e. αρσ = ασρ;

it is always possible, moreover, to make it diagonal by a proper choice of the

coordinate frame of reference.

If corrections for the so-called local field are negligible, it is possible to correlate the

two expressions 1.1 and 1.2 through the definition of polarization P as the average

dipole momentum multiplied by the number of molecules per unit of volume.

P = <p> N/V (1.15)

Only in particularly simplified cases, however (for example, a gas in standard

conditions of temperature and pressure [I.9]), it is possible to trace an easy

relationship between susceptibility and the polarizability tensor,

χ = αN/ ε0V (1.16)

where α is the scalar corresponding to the average of the elements αii of the

polarizability matrix in its diagonal form [I.10]. More generally, it can be said that

the electric susceptibility is the sum of the polarizabilities of the single molecules

contained in the unit volume, divided per the dielectric constant and the volume

itself.

Rayleigh and Raman scattering.

For the present description it is sufficient to refer to the induced dipole moment of

the molecule, and therefore, to the polarizability. This quantity is dependent on the

distribution of the electrons around the ions of the molecule; the perturbation

introduced by the incident electric field produces a displacement of the charge

distributions with respect to their position at the equilibrium: this phenomenon can

be expressed by the concept of an induced dipole. Polarizability depends also on the

characteristics of the exciting field: the response of the charge distribution is not

instantaneous [I.11], so the effect of applying a constant perturbation is not the same

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of an electric field which varies with a pulsation ω. In synthesis, from a

phenomenological viewpoint, it is possible to state that polarizability is a function of

the angular frequency of the electric field

p(ω) = α(ω)E(ω) (1.17)

for the simplest description of the elastic (or Rayleigh) scattering, that is of the

scattering of light at the same frequency of the exciting electromagnetic wave. The

actual form of this tensor can be derived by a series of descriptions, one of which is

represented by the Lorentz model of the atom as a harmonic oscillator with a

natural pulsation ω0.

More generally, the induced dipole possesses a spectral distribution which is not

simply a Dirac delta centered on the angular frequency of the exciting electric field.

This fact implies that the polarizability not only is a function of this variable, but it is

also relevantly influenced by the natural pulsations ωk of the vibrations of the

molecule. The induced dipole emitting at a generic angular frequency ωS, where S

stands for Scattering, can be described by the equation

p(ωS) = α(ω, ωk)E(ω) (1.18)

given vibration will hence induce a “modulation” of the linear optical polarizability

with a frequency equal to that of the internal vibration. Each of this motions can be

decomposed, as we have seen, in a basis of orthonormal vibrational coordinates Qk.

It is possible to expand in a Taylor series every matrix element of α according to the

normal coordinates of vibration Qk of the molecule and neglecting, for clarity of the

notation, the dependence on the pulsation of the exciting field. The expansion of the

polarizability component αij, where the indices i and j can vary among the chosen

reference axes, is

αij = αij,0 ∑k αij/ Qn)0 Qk + ½ ∑k,k’ 2αij/ Qk Qk’)QkQk’ (1.19),

stopping after the second order; the subscript 0 indicates that the derivatives are

evaluated at the origin of the frame of reference of the vibrational coordinates. The

electric harmonic approximation consists in arresting the expansion at the first

order, and here this approach is chosen. Since the expansion in power series is valid

for all components of the polarizability, equation 1.18 can be rewritten in vector

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form. If the perturbing electric field is a monochromatic plane wave of equation E =

E0 cos(ω0t), we obtain

p = αk,0E0 cos(ωt ∑k α/ Qk)0 Q0E0 cos(ωkt + k) cos(ω0t) =

= αk,0E0 cos(ωt ½ ∑k α/ Qk)0 Q0E0 cos((ω ± ωk)t + k)

Where the quantity α/ Qk)0 is called derived polarizability tensor or Raman

tensor of the normal mode k. It is apparent how, besides the presence of the so-

called Rayleigh component at ω, the emergence of two symmetric terms ω – ωk

(Stokes) and ω + ωk (anti-Stokes) per each vibrational mode is justified simply by

trigonometry [I.12]. The classical expression for Raman effect, therefore, is related

to the polarizability components which have a non-zero derivative with respect to a

molecular vibration at the equilibrium position of the molecule. The main

consequence of this fact is that not all vibrational modes can be investigated by the

detection of Raman scattering, but vibrations which correspond to a zero value for

the Raman tensor are said to be Raman-inactive.

The classical interpretation of Rayleigh and Raman scattering can, in conclusion, be

summarized as follows. If the molecule is at rest, the induced moment, and therefore

the scattered light, has the same frequency as the incident light. If, however, the

molecule is rotating or vibrating, this is not necessarily the case, because the

amplitude of the induced electric moment may depend on the orientation of the

molecule and the relative positions of its atoms. Since the configuration changes

periodically because of rotation and vibration, the scattered radiation is

''modulated" by the rotational and vibrational frequencies so that it consists of light

of frequencies equal to the sum and to the difference of the incident frequency and

the frequencies of the molecular motions, in addition to the incident frequency [I.7].

Another difference is that Rayleigh scattering happens always in phase with the

incident radiation, whereas Raman scattering bears an arbitrary phase relation due

to the phase k of the involved molecular vibrations [I.12].

(1.20)

24

Fig. I.2. Example of dependence of the polarizability from the vibrational modes and correspondent

Raman activity for a linear molecule of the type A-B-A (for example, CO2)[I.12]

I.3 Quantum theory of Raman scattering.

It has been shown that a classical theory of Raman scattering is possible, even if it

adopts a phenomenological viewpoint. Nonetheless, some of the conclusions which

can be drawn, for example, from equation 1.20 can be misleading. For instance,

correlating the Raman intensity (proportional to the square of the scattered field) to

the Raman tensor would predict equal intensities for the inelastic and superelastic

component of the spectrum, since the magnitude of the derivative of the

polarizability is the same for both the branches: prediction that is strongly denied by

the actual spectroscopic measurements, in which Stokes scattering is most easily

detected. Moreover, dependence of Raman on the vibrations of the molecule would

infer that no Raman spectrum can be obtained by molecules on the vibrational

ground state, contrarily to what is observed. This deficiency of the classical

treatment, in particular, is to be related to the absence of the zero-point energy in a

harmonic oscillator problem, which leads to the impossibility to describe

phenomena like spontaneous emission of light. A quantum treatment of the

25

scattering process is, therefore, necessary, even for a treatment that does not

include nonlinear phenomena such as hyper Raman scattering.

From a quantum viewpoint, Raman scattering may be treated as the inelastic

collision of an incident photon ħωo with a molecule on the initial energy level Enm

[I. 3]. Following the collision, a photon ħωs with different energy is detected and the

molecule is found on another energy level En. The energy difference ΔE = En-Em =

ħωnm, when positive, may appear as vibrational, rotational of electronic energy of

the molecule: this variation is called Stokes shift. If the molecule is initially in a

excited state, the difference can be negative and the superelastic photon scattering is

indicated as an anti-Stokes event. If the character of a vibration to be or not Raman

active can in principle be derived by classical or semi-classical description, the

intensity of a Raman band is not easily predicted by these treatments. For this

reason, a more correct formula for the Raman cross section of a vibration must be

the aim of the full quantum theory of light scattering.

Time dependent perturbations: the second order transition

probability.

In a quantum system described by the time independent Hamiltonian ℋ0, every

eigenstate is described by the symbol |u⟩ and corresponds to an eigenfucntion Ψu

and to an eigenvalue Eu: this eigenvalue governs the exponential time dependence of

the eigenfunction

Ψ ⟩ ⟩ e

/

Since the probability that the quantum system is in a state n is equal to the square

modulus of |Ψn⟩, it does not change with time: therefore, the description of any

transitions between two states must take into account the time dependence.

In Schrödinger’s representation the operators are constant whereas the wave

functions evolve with time. If a perturbation ℋi = ℋi(t) is introduced, so that ℋi(t)

Ŵ t > , the states u⟩ (from now on |u⟩), are still an orthonormal base for the

vector space. This means that a generic state |Ψ(t)⟩ can be expressed as

Ψ t ⟩ au t e i ut/ u⟩

u

(1.21)

(1.22)

26

provided that the complex coefficients au are functions of the time. As for the time-

independent case, their square modulus is proportional to the probability to find the

system in a state |n⟩ at the time t. If the initial state of the system is the state |m⟩, the

summation at time t = 0 is degenerate. Thanks to this property, an expression for an

can be obtained by substitution of 1.1 into the Schrödinger equation; this, after a

simple multiplication by ⟨n| eiEnt/ of both sides leads to

an

t

i au t e

– i u – n t/

u

n W u

Exact solution for this set of equations is possible only for limited cases (for

example, a two-level system). In general, it is necessary to proceed by successive

approximations by developing each coefficient au as a sum of different order terms.

au = au(0) + au(1) + au(2)

this approach is known as perturbative method. The validity of this method is

confirmed, in the case here discussed, as long as the applied electric field is a small

perturbation from the typical electric fields felt by electrons and atoms in molecules

(which is of the order of 1010 V/m)[ I.11]. A Raman scattering description must

consider at least to stop this expansion to the second order, because from a particle

viewpoint this phenomenon involves two photons. Since the perturbation is small,

the first order solution for a generic final state f is obtained by neglecting all terms

with u ≠ m in the equation . 3 they are strictly null at t , thus reducing the

summation to a single element

an

t

i e – i m – n t/ n W m

and integrating with time τ (with the consideration that the perturbing Hamiltonian

matrix has zero diagonal elements, i.e ⟨u Ŵ u⟩ = 0).

an

i e – i m – t/

τ

n W m dt

For higher orders, the procedure is iterative: the second order solution is given by

substituting into 1.2.2 the first order expression of the an. The resulting expression is

(1.24)

(1.25)

(1.26)

(1.23)

27

a

i a

t

e – – /

n W u dt

in which the expression of the first order solution can be made explicit. This

equation is valid for each choice of interacting Hamiltonian, provided that the

perturbation is “turned on” at time t . The electromagnetic field incident on the

molecule can be represented as a periodic potential e pressed by the function Ŵ t

Ŵ+ e–iωt Ŵ- eiωt, with Ŵ+ = Ŵ-† to make sure it is Hermitian and, hence, physically

meaningful. Spatial dependence of the electromagnetic field is thought as negligible

along the more significant dimension of the considered system, as it is for a molecule

exposed to NIR or visible light. If the energy difference between the state |n⟩ and |m⟩

is written as ħωmn, the expression for the first order coefficient is

a

n W m

e –

ω – ω n W m

e

ω ω

which is able to describe optical transitions where a single photon is involved

(absorption). The second order coefficient, substituting 1.28 into 1.27 and exploiting

the exponential dependence of the perturbing Hamiltonian with time, can be

synthetically written as

a

i

n W u u W m

ω ω

e

e dt

This expression can be separated in the sum of six summations: according to the

presence of one or the opposite sign, it is possible to distinguish different domains

for the energy values of the involved eigenstates. The sign of Ŵ is plus if n > Em,

minus vice versa, whereas the sign between the angular frequencies is chosen

according to the position of the intermediate energy level |u⟩: in particular, it is +

when Eu > Em. The transition probability Pmn is evaluated as the square modulus of

the coefficient an, which according to eq. 1.24 is given by the sum of the two

components an(1) and an(2),

Pmn ≈ an(1) + an(2) |2

This is the complete probability that a transition between the levels m and n,

mediated by an electromagnetic field represented by the perturbing Hamiltonian Ŵ,

occurs: all phenomena which involve one or two photons interacting with the

system (atom or molecule) are therefore included. Despite its generality, it is

(1.29)

(1.30)

(1.27)

(1.28)

28

already possible to highlight some peculiarities in the formula above reported. First

of all, the presence of differences at the denominators hints at the possibility of

indefinitely high value (in practice limited by the uncertainty at which the energy of

the quantum states can be measured) for the probability transitions: in other words,

the expression takes into account the resonant behavior which characterizes the

interaction of an electric field whose frequency is correspondent to a “jump”

between two states in the quantum system considered. Additionally, the transition

probability is shown to be dependent on the off-diagonal matrix elements of the

perturbing Hamiltonian Ŵ, which can be written as an integral quantity:

W u W v W d

This matrix expression reveals how the introduced perturbation determines which

transition between the unperturbed set of quantum states is or not allowed. A

theory of Raman scattering must start from a meaningful expression of the

interaction of the exciting light with the molecule or solid and then consider all the

terms inside 1.30 corresponding to inelastic (or superelastic) scattering, i.e. where

the angular frequency of the incident electric field is shifted.

A semi-classical expression for Raman effect.

This description treats the interaction of a quantum system (the molecule), whose

Hamiltonian H0 can be expressed as in equation 1.3, with a classical electromagnetic

field: the energy contribution of this interaction is represented by the symbol ℋi.

The interaction Hamiltonian for a multiparticle system exposed to an

electromagnetic field does not show an easy to handle expression, so it is often

necessary to approximate it with the so-called Electric-Dipole (ED) Hamiltonian:

ℋi = ℋED = – E p

which is by far the most important contribution to the perturbing Hamiltonian; the

other terms (magnetic dipole, electric quadrupole etc.) arising from the interaction

are negligible as far as ℋED is not zero, so in most cases the approximation is

(1.32)

(1.31)

29

excellent2. The electric field E is supposed to be a plane monochromatic wave of

angular frequency ω, whereas the operator p is defined as the sum of all the

coordinate vectors of the nuclei and electrons,

where q is equal to ±e if a proton or an electron charge is considered. For Raman

scattering of molecule this definition works also in a simpler depiction, with rj being

the atoms coordinates and q the effective charges they carry on. Sometimes,

moreover, the total dipole moment of the molecule can be considered as the sum of

biatomic dipole contributions [I.12] constituted by the couple of nearest neighbor

atoms. The vector components of p, px, py, pz are similarly defined with respect to the

projection of rj and r along the Cartesian coordinates.

The following treatment is a synthesis of the approach followed by Placzek [I.14]: his

description of the Raman scattering does not explicitly proceed through the

transition probability expression, but an analogous method is used to determine the

form of the electric polarizability. A transition dipole, equivalent to the matrix

element of the dipole operator among the time dependent wave functions Ψ’n, Ψ’n of

the perturbed system, is introduced

(p)mn = ⟨Ψ’n| p |Ψ’m⟩

This transition dipole can be considered as the quantum equivalent of the

oscillating electric dipole of the classic description. Thanks to perturbation theory,

an expression of the transition dipole in terms of the non-perturbed eigenstates |u⟩

can be recovered. By arresting the expansion of the perturbed functions to the first-

order, and after grouping all the terms with the same frequency dependence, the

expression of the real component of the component ρ of the vector p, with the choice

of the symbol ωmn = (En – Em)/ , results3:

2 For linear processes in E such as standard Raman scattering it is sufficient to consider the electric dipole term: for a standard molecular transition, the electric quadrupole and magnetic dipole terms, which follow in the expansion of ℋi, are of the order of the so-called fine structure constant α ≈ 1/137 [2]. 3 A more rigorous expression takes into account the decay time of each intermediate state |u⟩, which, differently from the initial and final ones, does not need to be stationary. Considering these decays as constants removes the divergence caused by the difference at the denominators.

(1.33)

(1.34)

30

p

n p u u p m

ω ω e

n p u u p m

ω ω

e

∑

e

e c. c.

where c.c. stands for complex conjugate; the expression, rather cumbersome,

manages to express the transition dipoles in terms of the matrix element of the

dipole operator, evaluated on the unperturbed states; the terms can be grossly

divided in two, the ones oscillating at the frequency sum and the ones at the

difference. The latter take into account the three kinds of scattering, provided that

the quantity ω – ωnm is positive: Rayleigh is obtained for |m⟩ = |n⟩; the terms with

the frequency (ω + ωnm) at the exponential, instead, do not correspond to any

scattering process. Recalling the classical expression which relates the induced

dipole to the electric field, it is possible to derive an equation defining the so-called

general transition polarizability (α)fi, whose components are of the form

α

n p u u p m

ω ω

n p u u p m

ω ω

Fig. I.3. Pictorial representation of the vibrational transitions corresponding to the three kinds of

scattering. For the anti-stokes transition the order of the n and m states is reversed with respect to

the notation adopted here.

(1.36)

(1.35)

31

In principle, the calculus of polarizability, and hence of the Raman scattering cross

section, is made possible only through an accurate estimation of every energy level,

wave function and lifetime of the molecule. Fortunately, a series of general

assumptions can be made to apply this formula to concrete cases of studies. If the

energy of the incident photon, for example, is not sufficient to excite the molecule in

its first electronic level, and the ground electronic state is not degenerate, the

eigenstates involved in the matrix elements can be identified with the set of the

vibrational eigenfunctions (vi). The transition polarizability, therefore, can be

written in the compact form

α v α v

Other simplifications stand in less favorable cases, introducing the coupling between

electronic and nuclear motion can be as a second perturbation. In any case, it is

possible in the end to come to a more manageable formula of this sort, for the

scattered intensity summed over all the space directions:

This form, much like the Raman tensor of the classic description, allows a rapid

determination of the so-called selection rules which determine if a vibrational

transition can be triggered by Raman scattering. In the mechanical and electric

approximation, the transitions between pure vibrational states are possible if, and

only if, the variation of the quantum number is ±1. A more general formulation of

this constraint regards the symmetry representations of the elements involved in

the equation of the transition polarizability. These components transform as the

binary products x2, y2, z2, xy, xz and yz. Group theory characters tables, therefore,

show which irreducible representation correspond to Raman or IR active mode

according to the presence of these functions.

Γ1⊂Γ(vm)Γ(αρσ)Γ(vn)

Where Γ1 is the totally symmetric representation [I.8, I.12]. This general rule makes

no statement as to the intensity with which a permitted transition will appear in the

(1.37)

(1.38)

(1.39)

32

Raman spectrum; in a mono-dimensional case it can be described as an argument

based on the parity of the functions which concur to the product inside the integral

(1.37).

Full quantum approach.

The full quantum approach becomes necessary whenever a resonance phenomenon

is affecting the Raman scattering [I.15]: it is through this formulation, indeed, that

the role of the coupling between the photons and the electronic states of the

material can be evaluated.

In a multi-level atom or molecule, several processes can occur in presence of an

incoming electromagnetic radiation (fig. I.4). In terms of the quantum theory of light,

Raman scattering consists in the destruction of an incoming photon (of frequency ν,

pulsation ω = 2πν and wave vector k, k= w n(ω)/c) in correspondence with the

creation of a photon of a difference pulsation ωs and wave vector ks: the process

leaves the quantum system on a different energy level and can be considered

instantaneous [I.16]. It is, as well, a coherent event [I.4], in the sense that the two

phases must satisfy conservation of energy and momentum: the differences ω – ωs, k

– ks, are therefore associated with a transfer of energy and momentum to the

molecule. This transfer results, in the second case, in the excitation of one or more

vibrational modes; if the scattering material is a crystal, the Raman scattering

corresponds to the excitation of a so-called phonon, i.e. a quantized oscillation

distributed along the whole lattice. The key difference between Raman scattering

and fluorescence is that in Raman scattering the incident photon is not fully

absorbed and instead perturbs the molecule exciting or de-exciting vibrational or

rotational energy states. Contrastingly, in fluorescence the photon is completely

absorbed causing the molecule to jump to a higher electronic state, and then the

emitted photon is due to the molecule’s decay back to a lower energy state [I. 7].

Three terms contribute to the transition rate associated with this event (creation

followed by destruction, destruction followed by creation, creation and destruction

simultaneously occurring) according to the quantum mechanical description: in this

formalism there is no difference between the order in which the two events occur,

provided that the energy conservation is satisfied by their combination [I.16]. Each

of these terms, indeed, is multiplied by a Dirac delta along the ωs function centered

33

at ω – ωmn, where ωmn is the pulsation of the final state. However, as already

emphasized, the energy ω does not necessarily correspond to any electronic

transition energy and the photon of energy ωmn is not absorbed in the strict

spectroscopic sense, as there is no conservation of energy in this stage of the

process. The role of the incident radiation is rather to perturb the molecule and

open the possibility of spectroscopic transitions other than direct absorption. If ω

approaches an electronic transition energy, enhancement of the scattered intensity

is observed [I.12]. It is important to note that Raman scattering is not the absorption

of the photon, followed by the emission of a photon of less energy, that instead

describes fluorescence.

Fig.I.4. A comparison between the most important different transitions involving the interaction of a molecule

with photons in the visible-near infrared range, on a schematic energy diagram [I.23].

Second quantization.

A step further in the quantum theory of scattering needs the introduction of proper

quantum operator for electric field. Let us consider for the moment only one mode

of oscillation. Electromagnetic radiation with a single frequency ωk and a wave

vector k is fully described by the potential vector Ak(t) = Ak e-i(ωt-kr) + Ak* ei(ωt-kr). It is

34

associated with an energy term which can be suitably expressed as the energy of a

classical harmonic oscillator if quantities

are introduced. The expression for the energy is, therefore,

2ε0AkAk*V = ½ (pk2 + qk2ω2). (1.41)

The quantum solution for this kind of problems requires the replacement of the

classical quantities with suitable operators qk q , pk

p , which can be considered as

a coordinate and its momentum, since the commutation relationship gives [p k,q k] =

– i . The Schrödinger equation for this kind of potential gives as a solution a set of

discrete eigenvalues for the radiation energy En,k = ωk (nk + ½), where nk is an

integer. For this reason, it is possible to consider each contribute of ωk as a

quantum of energy, called photon; in this way, to each eigenvalue for energy a wave

function called “number state” or Fock state and corresponding to the presence of n

photons can be found. This number states are eigenfunctions not only of the

Hamiltonian, but of the number operator n k = âk †âk, given by the product of the so

called operators “creation” and “destruction” whose definition is respectively

These two operators are not observables, but they correspond to the addition and

subtraction of one quantum of energy to the eigenstates of the Hamiltonian,

provided that a minimum amount of energy ( ωk/2, zero-point energy) is

conserved, transforming them from |nk⟩ to |nk+1⟩ and |nk-1⟩ respectively.

The set of all the possible modes of the electromagnetic field in a cavity (or in free

space) can be regarded as the cumulative wave function of all the number states.

|{nk}⟩ = |nk1⟩|nk2⟩

(1.40)

(1.40b)

(1.42)

(1.42b)

(1.43)

35

There is always an infinite number of oscillators, so the |{nk}⟩ form a complete set of

state for the electromagnetic field in the cavity.

Starting from these functions it is possible to recover the expression for the

Potential vector with straightforward substitutions, and hence the quantum

expression for electric and magnetic field. Equations 1.44 and 1.44b give the

expression for the k mode of both fields.

These expressions are used when the perturbation is added to the Hamiltonian of

the molecule. If the interaction between the incoming electromagnetic wave and the

molecule is limited to ℋED, then the dipole momentum gives rise to the transition

dipole pfi,

To make explicit the transition nature of this quantity, the dipole is associated to the

transition operator |i⟩⟨j|, which, when applied to |j⟩ gives |i⟩ and gives zero with all

the other eigenstates. Thanks to the closure theorem, the expression of the dipole

operator p can be rewritten in a more significant form.

The explicit form of the quantum operator p in ℋED is, then

⟩ ⟨ ⟩ ⟨ ⟩⟨

,

and therefore the perturbing Hamiltonian can be written as

ℋ

,

⟩

⟨

This e pression is responsible of the transitions between the states j and I, with I ≠ j

since the terms with I = j are null because of integral of odd functions. From the

(1.44)

(1.44b)

(1.45)

(1.46)

(1.47)

36

matrix elements it is possible to calculate absorption and emission rates for a

generic atom and photon distributions.

Before moving to the application of this formula to the study of the interaction of

particles with electromagnetic field, it is convenient to remind that the harmonic

oscillator problem is actually a common approximation for many physical problems.

The same vibrational energy contribution within the global molecular Hamiltonian,

as it was shown, can be expanded up to the second order and hence, each vibrational

degree of freedom Qk possesses an energy

Ev,k = (vk + ½) ωk, vk , , ,

where k, running from 1 to 3M-6, is the index of the vibrational modes and not a

wave vector. Vibrations, therefore, are ruled by the same quantum operators

introduced for photons, applied to the normal modes Qk: q k, q k†for the destruction

and the creation of a vibrational quantum respectively. Equivalently to quanta of

electromagnetic energy, quanta of vibrations can be treated as particles, which

statistically behave as bosons.

The Kramers-Heisenberg formula.

In principle, the forces between the atoms can be calculated a priori from the

electronic wave equation, but in practice this is not mathematically feasible (except

for H2), and it is necessary to postulate the forces in such a manner as to obtain

agreement with experiments. Therefore, although it is theoretically possible to start

with a model consisting of electrons and nuclei interacting by a Coulomb potential

and obeying the laws of quantum mechanics, in practice it is necessary to assume

the nature of the equilibrium configuration and of the forces between the atoms, so

that it seems more desirable to start with the model in which the atoms are the units

[20]. This system is then exposed to the perturbation of the electromagnetic field, in

the form of the quantum operators obtained by the second quantization process.

The following treatment allows the calculation of the cross-section for a multi-level

molecule by time-dependent perturbation theory. The hypotheses are the

assumption that the incident beam is weak enough to be considered a perturbing

element of the Hamiltonian, and that the broadening of the energy level is negligible.

(1.48)

37

Fig. I.5. Feynman diagrams of the two channels involved in the quantum description of Raman

scattering: the arrows describe the photons created or annihilated during the event, whereas on the

horizontal line the sequence of the molecule states involved in the transition are reported.

The transition rate (1/τ) for a process in which an incident photon of energy ω is

destroyed and another one of energy ωs is produced is calculated according to the

perturbation theory, arresting the expansion of the probability to the second order:

this quantity corresponds to the transition probability between two states m and n,

divided by time. The global transition rate for the scattering phenomenon is

summed over all the possible final states: moreover, in the first passages the

contribution of all the possibly involved phenomena are considered.

ℋ

ℋ ℋ

The expression contains both the first and the second order terms of the transition

probability and it is shown to be dependent only on the perturbation Hamiltonian

ℋi = ℋED. The first term inside the modulus reproduces the Fermi golden rule

expression and provides the rate of first-order transition form m to n; the second

(1.49)

38

term provides the indirect transitions from state m to state n via a range of virtual

intermediate state u [I.16].

A general scattering event does not involve a single mode of the radiation field, but

the creation and the destruction operator affect different modes of the

electromagnetic field: thus, it is necessary to introduce â†, â, â†s,âs. A quantum state

describing the interacting system is indicated by the bra |l, ls, i> where the first two

letters are the number of photons of the incident and the scattering fields

respectively, while the third indicates the molecule energy level. Adapting the

equation 1.49 for the transition rate to new notation, the expression for an initial

state of l incident photons and the molecule in the ground state (m = 0) results4 in

, , ℋ , , ℋ , ,

This kind of equation belongs to the class of integral equations that give the

solutions for quantum mechanical problems of scattering theory [I.18]. The second-

order transition rate can be simplified and a more explicit expression shows the two

concurring paths for the interaction of the two photons:

τ

πe ωωs3l

s nu um

ωu ω

nu s um

ωn ωu

ω ωs ωnm

ksn

where , s are the unit polarization vectors of the incident and scattered photons

and Dnm is the matrix element m|D|m . This expression correctly predict the

dependence on the third power of the scattered pulsation, which, multiplied by the

angular frequency of the incident wave, recover the fourth power typical of the

4 A more rigorous form can be found in the 2nd edition of Loudon [I.16]: the presence of the nonlinear Hamiltonian gives rise to an additional term inside the square modulus. The first term at the right hand side of the equation corresponds to a non linear event which destroys a photon k and creates ks: the Hamiltonian in this case is proportional to A2[5]. The second term (proportional to Ap) is implicitly made out of two contributes which differ in the type of the intermediate state l: one produces the operators in the order âs

†â, the other â, âs†. Separately the two contributes do not

conserve the energy, but their combination does, so can it be accepted; in the time between the two events the molecule belongs to a state l which is different from both the initial and the final state. Due to the relationship B2 = E2/c2, the nonlinear term can be neglected in most cases (when the denominator in the second term is different from zero).

(1.50)

(1.51)

39

scattering events.The transition rate can be converted to a cross-section by dividing

it for the flux density c/V

This expression, which was obtained first by Kramers and Heisenberg, is suitable for

describing both Rayleigh (m = n, and Thomson, for high frequency) and Raman

scattering. The summation is limited to ωf < ω so that the delta function is not

needed but the conservation of energy requirement is automatically fulfilled.

Complete formula takes into account the two independent photon polarizations

[I.16]. Both this equation and the semi-classical formula obtained by Placzek [I.14]

provide a general framework, and can be simplified according to the range of

energies involved by the scattering, or to the kind of molecular functions involved

(purely vibrational, vibronic, etc.).

General considerations.

- Raman scattering is much less effective than the elastic scattering, which normally

regards a small fraction (10-3, Raman scattering Theory, David W. Hahn) of the

incoming photons. Thus, high intensity light source are required for observing it.

Another strict constraint is, naturally, the monochromatic character of the exciting

electric field. For best resolution performances, a narrow emission-line laser is the

choice of preference.

- Raman spectra provide the same kind of information of IR absorption spectra, but

they obey to different selection rules: therefore, in many cases the two techniques

work in a complementary way to characterize the structural and chemical

properties of a material.

- Stokes branch is more intense than anti-Stokes: the main reason is

thermodynamical, and a proper evaluation of the ratio between symmetric bands

can be obtained with the quantum mechanic treatment.

- Vibrational spectrum are generally reported in terms of the relative shift,

expressed in wavenumbers or inverse wavelengths, between the energy of the

detected photon and the one of the exciting photon. The most common unit for the

inverse wavelength coordinate is the cm-1. This allows an easy comparison not only

(1.52)

40

among spectra of the same method obtained with different excitation wavelengths,

but also between Infrared and Raman spectra of the same material, despite

corresponding to different techniques, can be put side by side. On the other hand,

one must not forget that the spectral extension of the spectra is, in the absolute

frequencies scale, much different, with evident consequences on the potential

resonances effect of the chosen source of radiation and on the sensitivity of the

detector. If, for example, the N2 vibrational mode is considered, corresponding to

2331 cm-1, this will be found at 387 nm if the exciting wavelength is 355 nm; if,

instead, the incident wavelength is 632 nm, the mode will be shifted at 741nm [I.19].

- Even if only a full reconstruction of the vibrational levels of the molecule can

provide the correct description of every spectral feature, in many practical uses

Raman spectroscopy can be set aside by an extensive approach. In many molecules,

indeed, atoms can be grouped in sub-units which often possess their own vibrational

modes that are not strongly affected by the global geometry of the molecule. On the

basis of this consideration, it is possible to report in tables, now available for many

class of materials, the typical energy interval in which the vibrational mode

involving the sub-group can be found. An example is shown in figure I.6.[ I.3]

- Since Raman spectroscopy traditional probes use light sources in the visible or

near infrared wavelength range, Raman scattering is not the only form of radiation-

molecule interaction. On the contrary, it is much likely that the material shows

absorption for the chosen wavelength, and therefore luminescence (excitation of

electronic upper energy levels, followed by a radiative decay) is often a competitive

phenomenon for Raman. Different approaches have been proposed and adopted to

overcome, or at least to limit this problem: the last paragraph of this chapter

describes Surface-Enhanced Raman Spectroscopy as an increasingly convenient

option in this direction.

- The treatment reported here below does not consider the angular dependence of a

Raman scattering event. While useful consideration could be drawn from this

description, introducing the formalism needed for this part of the problem will

exceed the scope of this thesis. We refer to Griffiths [I.20] and to Sakurai [I.21] for a

complete theory of scattering.

41

Fig. I.6. Energy distribution of the most common vibrational modes in the spectral region 0-700 cm-1

[I.3].

I.4 The cross section.

Experimentally, the quantity that can be revealed by a detector of electromagnetic

radiation is the intensity; it is convenient, therefore, to quantify the probability that

a Raman event takes place not in terms of the scattered field, but of its square

modulus. Indeed, the classical expression [I.10] for the intensity associated to a

radiating dipole, along a direction which creates with the dipole p an angle ϑ is

IS = ωS4 |p|2 sin2ϑ /(32 π2 ε0 c3)

Where the temporal variation of p is averaged over an oscillation cycle. This quantity

can be also written in terms of the scattering direction, represented by the unit

vector eS

(1.53)

42

IS = ωS4 |eSp|2 /(32 π2 ε0 c3)

There is a way to correlate the intensity of the scattered field with the one incident

on the area of interest: cross section (σ) gives at a glance an indication on the

efficiency of the considered scattering process. Loudon defines σ as the rate at which

energy is removed from the incident beam by scattering, divided by the rate at

which the energy in the incident photon beam crosses a unit area perpendicular to

its propagation direction [I.16].

σ ωs

ωI Isr

d

In case of Raman scattering, the value of σ is not only related to the exciting laser

wavelength, but to a particular vibrational mode of the selected molecule of angular

frequency ω – ωs. This quantity can be expressed in terms of the transition rate τ of

a scattering event, provided that the volume V which participates to the scattering is

well defined as the product of the cross section times the length crossed in that time

by the light in the material, nk c τ.

A differential cross section, dσ/d can be introduced when the cross section angular

distribution is of interest: this is often the case for an electric dipole, where the

scattered radiation is not isotropic. A possible definition for the differential

scattering cross section is given by Cardona, as the ratio between the energy emitted

per unit time by an electric dipole M and the energy incident on that dipole divided

per unit area and unit time, [I.22]

dW = d |es α el|2 EL2ω4/(4π)2 ε0c3

by the energy incident on that dipole per unit area and unit time, W = ε0c EL2, and

integrating over the solid angle ; this definition can be proven to be equivalent to

the. The unitary vectors el and es respectively refer to the polarization of incoming

and scattered of (laser) light. The expansion of the polarizability carried out in 1.5

easily leads to an expression for the Raman component of the scattering cross

section. In equation I.56 the differential cross section for a Stokes-shifted line at

angular frequency ωs = ω0 – ωk is reported. [I.5, I.11]

dσs/d = |es α/ Qn el|2<QkQk*> ωs4/(4πε0)2c4

where < > represents the thermodynamical average over the ground state of the

molecule [I.22], needed to take into account the different population of the

(1.54)

(1.54)

(1.55)

(1.56)

43

vibrational levels in an ensemble of many molecules. This formula closely resembles

the intensity formula (1.37) obtained by the semi classical theory, and reveals the

explicit dependence of the polarizability tensor on the normal coordinates Qk.

I.5 Surface Enhanced Raman Scattering.

Though Raman spectroscopy gives detailed chemical and conformational

information, the small scattering cross-section has limited widespread use.

Experiments performed in the mid 1970s demonstrated that molecules adsorbed to

a roughened metal surface generated anomalously large Raman intensities [21]. This

phenomenon is known as Surface-Enhanced Raman Scattering (SERS), where the

last S can also be used to refer to Spectroscopy, where the attention is given to the

technique more than to the effect exploited. Enhancements varying from 103 to

1011[I.23] have been reported for molecules adsorbed to nano-structured silver,

gold and copper, in order of width of use, but other metals have found application in

minor cases. While there is still occasion to debate about a commonly accepted

theory of the phenomenon, general agreement is given to the attribution of the main

reason for this boost in the Raman cross section to a so-called Electromagnetic

Mechanism (EM). Occasionally, this factor can be helped by a Chemical Mechanism

(CHEM), which nonetheless is of lesser importance.

Electromagnetic Mechanism is mostly related to the excitation of a localized surface

Plasmon resonance by the laser source. Briefly, the nanometric features of the metal

substrate on which the molecule is adsorbed are characterized by collective

oscillations of charges (typically conduction electrons), which can be selectively

e cited by resonant laser wavelengths. These “plasma waves” are not only localized

inside the roughness features of the conductor, but naturally they are confined to its

surface: hence comes the name of Surface Plasmon Polaritons. This allows the

Raman cross section of the modes of the adsorbed material, which are parallel to the

excited plasmons, to benefit of the huge electric field which is localized along the

metal surface. CHEM enhancement, instead, is not only site-specific but it also

depends on the analyte: it is present when a charge-transfer state is created

between the metal and the adsorbed molecule. If the laser wavelength is not only in

resonance conditions with the metal nano-features, but with an electronic transition

44

of the molecule under analysis, the effect experiences a further enhancement and it

is called SERRS, where the first R of the acronym stands for Resonance.

The character of resonance technique is, however, implicit in the description of the

Surface-Enhanced Raman effect: for this reason, even if the enormous gain given to

the signal to noise ratio of the vibrational spectrum makes this technique preferable

for samples in very low concentrations, or for cases in which fluorescence would be

an unavoidable obstacle, some caveats must be considered whenever a SER analysis

is planned. First of all, the enhancement provided by the interaction with the

plasmon is naturally very selective: not all the vibrational modes are affected in the

same way, but, as previously reported, geometry plays a fundamental role in

determining the effect, due to the relative orientations of the vibrational modes with

the excited plasmons, the polarization of the incoming field, the distance between

the roughened surface and the different parts of the molecule, etc. Without going too

much into details, these variables result in a distortion of the SER spectra with

respect to the normal Raman ones, which can be traced in terms of a different

intensity ratioes between the peaks or, if the local environment of the molecule has

significantly changed, in broadening or shifts of the vibrational features. It must be

noted, moreover, that there does not exist a perfect SER substrate, which can be

used for every sample, but different materials and shapes produce variable effects

according to the wavelength of the chosen laser and, of course, to the analyte.

Nonetheless, the study and the exploitation of SERS are growing: in the last ten years

the number of published works dedicated to or containing the SER technique more

than doubled, and the trend does not seem to decrease [I.24]. The development and

consequently the availability of cutting-edge methods permits the researcher to

adopt an increasing number of possible geometries, shapes of the structures,

materials and, in general, probing methods. If the stability still makes the traditional

silver or gold nanoparticles a reliable starting point for a SER measurement, the

current tendency is to design the substrate parameters for a particular analyte,

making SERS a truly tailored detection and identification method.

45

Bibliography.

[I.1] A. Smekal, Naturwiss. 16, 873 (1923);

[I.2] C.V. Raman and K.S. Krishnan, Nature, 121, 501 (1928);

[I.3] E. Smith, G. Dent, Modern Raman spectroscopy: a practical approach, John Wiley

& Sons(2005);

[I.4] I.R. Lewis, H.W. Edwards. Handbook of Raman Spectroscopy. From the Research

Laboratory for the Process Line, CRC Press (2001);

[I.5] R. Aroca, Surface Enhanced Vibrational Spectroscopy, John Wiley & Sons

(2007);

[I.6] N. Ashcroft, M. David , Solid State Physics, Cengage Learning Emea (2000);

[I.7] E.B. Wilson, J.C. Decius, P.C. Cross, Molecular Vibrations: The Theory of Infrared

and Raman Vibrational Spectra, Dover Publications (1955);

[I.8] D.C. Harris, M.C. Bertolucci, Symmetry and spectroscopy: An Introduction To

Vibrational and Electronic Scattering, Dover Publications (1989);

[I.9] G.Moruzzi, Dispense di struttura della materia. University of Pisa

[I.10] P. Mazzoldi, M. Nigro, C. Voci, Fisica – Volume 2: Elettromagnetismo – Onde,

Edises (2000);

[I.11] E. Le Ru, P. Etchegoin, Principles of Surface Enhanced Raman Spectroscopy

and related plasmonic effect, Elsevier Science (2008);

[I.12] D. A. Long, The Raman effect. A unified treatment of the theory of Raman

scattering by molecules, John Wiley & Sons (2002);

[I.13] W. Demtröder, Laser Spectroscopy - Basic concepts and instrumentation,

Springer (1996);

46

[I.14] Placzek, G. Rayleigh-Streuung und Raman-Effekt, in Handbuch der Radiologie,

E. Marx (ed.), 6, 205–374. Academische Verlag: Leipzig (1934);

[I.15]W. H. Weber, R. Merlin, Raman Spectroscopy in Materials Science, Springer

(2000);

[I.16]R. Loudon, The quantum theory of light. Second edition, Clarendon Press,

Oxford,(1983);

[I.17]H.A. Szymanski, Raman Spectroscopy: Theory and Practice, Plenum Press,

(1967);

[I.18] E. Merzbacher, Quantum mechanics, 3rd edition, John Wiley & Sons (1998);

[I.19] David W. Hahn, Raman Scattering Theory, University of Florida (2007);

[I.20] D. Griffiths, Introduction to Quantum Mechanics, Prentice Hall, (1995);

[I.21] J. J. Sakurai, Modern Quantum Mechanics, Yoshioka Shoten, Kyoto, (1989);

[I.22] M. Cardona, G. Guntherodt, Light Scattering in Solids, Vol. 2, Basic concepts

and instrumentation, 3rd edition, Springer (1989);

[I.23] R. P. Van Duyne, C. Haynes. Raman Spectroscopy, Northwestern University

(review available online);

[I.24] B. Sharma, R. R Frontiera, A.I. Henry, E. Ringe, R. P. Van Duyne, SERS:

Materials, Applications, and the Future Surface Enhanced Raman Spectroscopy,

Materials Today, 15 (2012), 16–25.

47

CHAPTER II

THE REMOTE RAMAN SCANNER.

The determination of the physical processes resulting in a Raman scattering event,

carried out in the first chapter, enables the researchers to consider all the necessary

parameters in the preparation of an experiment. It is even more crucial, when

designing a novel instrument which relies on this effect, to determine the physical

quantities it involves, and hence the most suitable techniques for the detection of

this precious but weak signal, the critical points and the advantages of the possible

approaches and the recent discoveries and innovative methods concerning Raman

spectroscopy. This chapter focuses on the development of the Remote Raman

Scanner instrument, starting from its objectives to the phases of the project to the

construction. An introductory section, however, is dedicated to the application of

Raman spectroscopy to the field of Cultural heritage, in order to appreciate in which

direction the proposed device should be situated.

II.1. Raman spectroscopy in Cultural Heritage.

Identification of materials in and on objects of cultural and artistic interest has long

been recognized as a task necessary for uncovering the history of such objects and

finding out possibly how, where and when they were made, what was their use and

even how they have interacted with their environment during their lifetime.

Furthermore, knowing the materials not only opens a window to the past for

archaeologists and historians but also enables conservators to pave a safe way to the

future by means of proper preservation [II.1]. The main challenge in this type of

work is the vast diversity and variety of materials encountered, which include:

pigments, mineral or organic, natural or synthetic; metals or metal alloys and their

corrosion products; stone and glass; bio-organic materials such as wood, leather,

48

paper, parchment, but also oil, protein or sugar-based binding media and glues and

synthetic or natural varnish coatings and associated by-products arising during their

aging [II.2]. In this context, Raman spectroscopy proves to be an effective option,

providing a deep insight on the molecular [II.3] and structural [II.4] properties of a

material without altering or damaging it. As an optical measurement, it does not

need any contact between the probe and the sample and the stimulation of scattered

light does not imply high light intensities, so the power of the exciting

electromagnetic field can be kept well below the threshold of polymerization,

ablation or burning, with respect to the possible risks concerning different

materials. The detection of highly specific fingerprints such as the frequencies of the

vibrational modes enables the researchers to distinguish among similar materials

and even among different crystal phases of the same compound, such as in the case

of minerals; moreover, the vibrational frequencies depend on the strain undergone

by a solid sample, so they can be used as a stress indicator (showing deformations

before they become irreversible); furthermore, the influence of thermal energy on

the ratio between the Stokes and anti-Stokes spectral branches, can be measured to

monitor the temperature evolution of a sample, for example due to exposure to the

laser beam [II.5].

A huge number of publications attests in the last 20 years the widespread

possibilities of employing Raman spectroscopy on objects of art such as paintings

[II.6], frescoes [II.7], drawings [II.8], mosaics [II.9], sculptures [II.10], textiles [II.11],

illuminated manuscripts [II.12], old prints [II.13], parchment [II.14], miniatures

[II.15], but also on pottery [II.16], porcelains [II.17], coins [II.18], jewels [II.19],

human and even feline mummies [II.20,II.21]! ssessment of an artwork’s

conservation state and of the validity of its restoration; or interpretation, dating and

etiology of a controversial artifact; these are all examples of case studies which

benefit of this incredible source of information.

Of course, Raman spectroscopy alone is not generally sufficient to provide a

evidence to every identification problem, but it is usually employed together with

other non-invasive (IR spectroscopy, fluorescence spectroscopy, X-ray spectroscopy,

etc.) or micro-invasive methods (laser-induced breakdown spectroscopy, gas

chromatography-mass spectrometry) in order to obtain complementary

information. As explained in the previous chapter, indeed, Raman spectroscopy is

49

based on a rather weak effect, and detection of the materials’ spectral fingerprints is

often hampered by the presence of a high fluorescence background signal. This is

particularly true when the sample is of biological origin, like binding media,

varnishes and dyes in the application to artworks. Raman spectroscopy, however,

has several advantages (rapidity, possibility to be performed in situ, no need for

sample preparation) which frequently make it preferable even to more “efficient”

techniques: in particular, because of these features it is an ideal preliminary method,

useful to restrict the range of possible answers to an identification, before more

tailored, but possibly more invasive experimental techniques are considered.

II.2. The aim of the project.

The proposed instrument must conjugate some of the most important features for

the analysis of valuable (and sensitive) objects. It is going to be operated, indeed, by

a research group whose primary activity is the study of objects of artistic or

archaeological interest [II.22], together with other already available techniques

[II.23-II.25]. Raman spectroscopy, as previously stated, is one of the favored

techniques to collect molecular information from this kind of materials.

In such delicate applications it is particularly relevant that the analysis method is

non-invasive: this requirement is more restrictive than non destructivity, since it

implies that the experiment does not affect the integrity of the sample and possibly

does not interfere with the ambient conditions in which it is kept [II.26]; for Raman

spectroscopy, this necessity often means that the contact or proximity approach are

not enough, but in many cases a certain distance between the instrument and the

analyzed surface is required to preserve the physical, chemical or biological

environment of the object of interest (e.g., a fresco in a narrow niche, an ancient

scroll under a controlled atmosphere, a mural frieze in a cave).

Remote analysis, in addition, can be integrated with another desirable capability for

the instrument: the possibility to analyze an extended area of the target surface

without moving the sample. In the study of Cultural heritage, indeed, a punctual

analysis is rarely significant for the entire object, and therefore a meaningful Raman

analysis implies either to extract several samples from the studied item, or to shift

the Raman probe at different positions over its surface. Such a feature, therefore,

50

would be essential, not only to expand the sampled area of a target material, but it

could also foresee application on bulky or precious artifacts for which analysis is

often prevented by practical impediments connected to the impossibility of being

moved.

This requirement is directly related to the third one, which is the transportability.

The already mentioned reasons are driving forces towards an instrument with

compact size and limited weight, which could be easily carried in a museum, a

cathedral or an archaeological site. The planned Raman instrument is accordingly

designed to work as a transportable remote imaging spectrometer: the imaging

method, as explained in the next sections, involves the acquisition of a Raman

spectrum from different points of a surface, and the spectra are combined together

after the measurement.

Before engaging in the project of a system, though, it was important to explore and

compare the range of solutions offered by already existing devices, commercial or

developed by other research groups, designed for similar purposes. A brief report of

the available techniques of Raman imaging, remote and in situ Raman analysis is

given in the next paragraph, with a particular attention for applications in the

Cultural heritage field.

II.3. The state of the art.

Raman imaging.

Raman imaging can be implemented with two different approaches: direct Raman

imaging and Raman mapping. In direct Raman imaging, the laser spot is defocused in

order to illuminate a relatively large sample area, and an image detector, coupled

with suitable filters, is used to acquire images at specific Raman shift frequencies (or

wavenumbers). For the purpose, dielectric [II.27, II.28] and acousto-optic or liquid

crystal filters [II.29-32] are most often employed. The use of a single dielectric filter

integrated with a tunable laser has also been proposed [II.33]. The direct approach

has the great advantage of being faster than Raman mapping due to the parallel

acquisition provided by a wide field imaging detector. Nevertheless, the intensity of

51

the collected Raman signal can be very low due to the need for defocusing the laser

beam and to the attenuation factor of filters. As a consequence, direct Raman

imaging is typically limited to microscopy applications, the width of the field of view

being of hundreds of microns, and is especially useful when a priori information on

the analyzed surface is already available, allowing one to investigate few selected

spectral Raman bands [II.34]. In contrast, in the Raman mapping approach a Raman

image is generated by performing a serial acquisition of many points in the area of

interest. With this method, when the laser is focused on a spot (point mapping), the

information is reconstructed by sequential single-point acquisitions at each x, y

position with a suitable Raman spectrometer. In contrast, with line mapping the

laser is focused onto a line and a bi-dimensional detector, coupled with a dispersive

element, is used to register both the spatial and the spectral information of the

Raman signal produced along the line [II.35]. Generally, the line mapping approach

is preferable to the point mapping approach, thanks to a lower acquisition time and

higher SNR.

An algorithm introduced by Puppels et al. [II.28], estimate the signal to noise ratio

for both Raman imaging and mapping, and therefore helps to determine the best

option with respect to the theoretical experimental parameters (exposure times,

number of pixels of the detector, readout noise): this method, however, is hardly

applicable to real case studies, because of the non-uniform frequency response of

the filters and the detectors, and the difficulty in quantifying the terms introducing

losses in the signal collection.

For the analysis of art, Raman mapping proved to be particularly advantageous,

since it provides high spectral resolution. For example, recent works have

highlighted the capability of Raman mapping to analyze polished cross sections of

paint layers [II.36,37], to evaluate the penetration depth of inorganic and organic

substances in conservative treatment [II.38], as well as to study the yellowing effect

of laser cleaning treatments for the removal of black crusts from stones [II.39]. In all

the reported cases, however, the size of the analyzed area is never bigger than

1 mm2[II.37, II.40].

Remote Raman sensing.

52

Here, and in the rest of this work, the term “remote” is adopted to indicate

instruments which are kept at a certain distance from the sample under

investigation, and not to the devices which can be controlled by far, such as the

Raman spectrometers for oceanographic studies [II.41]. The former, which are also

called stand-off spectrometers, usually perform Raman spectroscopy at a distance of

the order of tenths of meters [II.42]. If its principle was first proposed in the mid-

1960s, only thirty years later the technology was mature enough to permit the first

measurements, carried out on atmospheric phenomena [II.43]. Ranged

measurement are generally favorable over proximity analysis because, when

performed in situ, they suffer less from vibrations and other stability issues. of

course increasing the working distance [II.44]. Naturally, the increased focal length

generally implies a lower acceptance angle for the collection optics, and as a

consequence the signal intensity is considerably reduced. Nonetheless, the adoption

of powerful solid-state laser sources (preferentially frequency-doubled Nd-Yag,

[II.45]), typically working in pulsed regime, is sufficient to provide qualitatively

good spectra from samples which do not risk to be damaged by high energy pulses.

The collection of the scattered light exploits casse-grain telescope objectives, which

can be directly coupled to spectrograph or to acousto-optic tunable filters (AOTF),

or connected by optical fibers to the detection apparatus. One of the reported

devices is also able to work in a scanning mode, thanks to a two-axis motorized

motion of the prisms which deliver the laser beam inside the telescope [II.45], still

no evidence of the effectiveness of this approach has been published yet.

These instruments, however, are generally conceived for the remote analysis of

dangerous materials such as explosives [II.46], or samples in difficultly reachable

positions, like rocks or even clouds. Their working distance is at least of the order of

the tenths of meters (it has been driven up to 533 m [II.45]), and therefore they find

little application to the study of Cultural heritage. Currently, no intermediately

ranged instrument is available, neither as a commercial device, nor as a one-off tool

built by a research team. A short range approach, however, has been chosen by

Vandenabeele et al. [II.47] for the design of the first Cultural-Heritage-oriented

Raman spectrometer, called MArtA (Mobile Art Analyzer): the instrument is

portable, and its working distance can be adjusted up to 2.5 cm, settling it out of the

standard micro-Raman set-ups. A more thorough report about its performances is

53

given in the following section; its focal length, however, can be considered to

determine the order of magnitude of the longer working distance currently available

for a Raman instrument not based on a telescope reflector.

Portable Raman instruments.

In the last ten years, the availability of robust, air-cooled laser sources, the

miniaturization of detectors and their electronics as well as the lap-top computer

increase have led progressively to the development and use of more and more

compact Raman set-ups: transportable, at first, then mobile (< 30 kg) and even

hand-on (< 2 kg at least for the remote head) ultramobile spectrometers are now

available [II.48]. Generally speaking, laboratory equipment still performs better than

mobile Raman instrumentation. It is clear that during the design of the mobile

instrument some concessions have been made in order to create a compact and

stable Raman spectrometer. Besides, non-mobile dispersive Raman instruments are

usually equipped with multiple lasers, allowing the researcher to select the most

appropriate laser wavelength for the investigation on hand. Moreover, laboratory

instrumentation is in general well positioned in a dark air conditioned room on a

solid laboratory table, guaranteeing optimal alignment conditions and a high

throughput of light [II.48]. Nonetheless, industrial manufacturers and specialized

research groups spend many efforts in the development of increasingly effective

portable Raman spectrometers, because the potential fields of application are

enormous: not only Cultural heritage, but also geosciences [II.49] and forensic

science [II.50], just to cite three of the sciences which have already started to benefit

of these instrumentations.

Among the stand-off instruments, the prototype built by Vandenabeele, MArtA,

which exploits a 785 nm diode laser was cited. This instrument, in which a probe

made out of a bundle of 37 optical fibers is equipped with a interchangeable focusing

lens, the focus being helped by remote-controlled micro-positioners [II.51]. MArtA

proved to be efficient over quite a wide range of materials, from Egyptian sarcophagi

to paintings on paper or wood, to mediaeval frescoes [II.51] a comparison with

mobile commercially-available Raman spectrometers has been carried on by

Vandenabeele itself, with a positive validation [II.52]. Another portable Raman

device was adopted by C. Miliani et al., and is one of the numerous analytical

54

methods available on the Mobile Laboratory (MOLAB) of the Perugian research

group [II.53]: this is however, one of the first commercially available instruments, a

Jasco RMP-100 microprobe, equipped with an Olympus objective (50× or 20×), for

a total weight of 25 kg. This instrument allows measumrement of areas of the order

of 5 × 5 µm2 approximately [II.53]. A completely different approach has been

followed by R. Ernst for the development of his spectrometer, based on a Senterra

Raman microscope which is mounted on a transportable gantry: this frame supports

the microscope probe, allowing it to be translated over large flat samples (up to 100

× 70 cm2). This instrument has been successfully applied in situ for a series of

measurement on Mongolian thangkas [II.44] and it is surely very suitable for

monitoring wide objects such as paintings, but it cannot perform analysis without

removing them from their original vertical position. Moreover, its structure does not

allow analysis of three-dimensional objects: the vertical translation stage allows

very precise focusing but it does not permit wide excursions from the default

distance .

Since the first pioneering attempts, new portable Raman spectrometers have been

introduced by the the researchers and by the manufacturers, resulting in a wide

range of commercial and custom instruments which are finding an extending

application also in different fields from archaeometry and conservation. For

example, a challenging and growing potential application of this technique is to take

part in a future mission to Mars.

The preferred choice for the laser sources used by these devices is the diode laser

emitting at 785 nm, closely followed by the Neodimium: Yttrium Aluminum Garnet

(Nd-YAG) frequency doubled at 532 nm: these two options, indeed, offer relatively

cheap solutions, with the significant advantage of a robust and not-cumbersome

packaging.

The great majority of the reported mobile devices are able to perform meaningful

spectral analysis in a dark environment, because they suffer from stray light noise:

therefore, either they require the room which hosts the object of interest to be

obscured, or, for instrument with a compact probe head, they need to apply a black

coat around the analyzed area to screen the surrounding illumination.

55

II.4. Remote Raman Scanner: the layout.

In the application to artworks and archaeological finds, thus, the mainstream is to

perform Raman measurements with a mobile instrument, which relies on a

traditional microscope layout or is equipped with a fiber connected probe, whose

working distance is of the order of tenths of millimeters in the best case. All the

reported instruments, consequently, suffer from instable supports: vibrations occur

very frequently when the measurement is performed in situ, and as a result the non-

perfect focus of the laser beam affects the quality of the spectra. This issue must be

generally assigned to the difficulty in reaching the desired area of the object of

interest in environments that have not been built according to this purpose.

Insensitivity to slight focus variations and a longer working distance can actually

help the on-site monitoring of Cultural Heritage, by reducing the set-up times for

the measurements, but, most of all, enabling the analysis of otherwise off-limits

specimens.

For this reasons, the project choice for the Remote Raman scanner was to design a

stand-off spectrometer able to work at an intermediate distance between traditional

Raman devices and the spectrometers for the remote analysis; therefore, it does not

rely on a microscope objective or a casse-grain layout, but on a custom lens-built

optics with a focal distance of at least 20 centimeters [II.54]. The capability of

performing a Raman analysis of a wide area is obtained thanks to two factors: the

field of view is widened by the choice of a long-working-distance optic, and the

selection of the focused point within the field of view is performed by a deflection of

the laser beam. The last feature is achieved by inserting in the light path a couple of

orthogonal galvanometric mirrors, which tilt of an arbitrary angle the reflected

beam (within the allowed range) respectively along the x and the y Cartesian axis of

the focal plane. The presence of these two additional degrees of freedom in the light

path introduces the task of determining the suitable optics to correctly focus the

laser beam at the chosen focal distance for any angular shift provided by the

mirrors. This requirement would not be particularly challenging, if the same optics

weren’t used for the collection of the scattered light as well. The geometrical

configuration selected for the device is, indeed, indicated as “backscattering mode”:

the optical axes referred to the beam, before and after the scattering event with the

56

target material, are disposed with an angle of pi (180°) with respect to each other.

Naturally, this implies that the optics which is responsible for the collection of the

Raman signal must be the same which focuses the excitation laser beam. More

details on the optical system are reported in a dedicated paragraph.

The design of the instrument determined other relevant choices for the project: a

semiconductor laser, with an external cavity configuration, emitting at 785 nm, is

used as the light source; a cooled Charged Coupled Device (CCD) sensor is adopted

as the detector of the scattered light; dispersion of the spectral components of the

Raman signal is provided by a single-turret Czerny-Turner spectrograph, while

rejection of the elastic scattered light is assured by a dichroic filter and a notch filter,

centered at the laser emission wavelength; connection of the laser source with the

optical system, and from the latter to the dispersing element and the sensor, relies

on optical fibers, coupled with collimating lenses. Minor specifications about the

instrument set-up are given in the next sections: it is worth noting, however, that a

consistent part of the project has been the development of a specifically-thought

unified software control, which provides the interface for the user of the Remote

Raman Scanner in order to manage the available experimental parameters and to

monitor and save the results of the analysis.

The definitive version of the instrument can be seen in figure II.1: the laser source

and the detection stage are lodged on a dedicated scaffold (transportable by a

trolley), and two optical fibers (3-meters long) are used for connecting them with

the proper probe head, built on a carbon fiber reinforced plastic breadboard and

mounted on a photography tripod. This part of the instrument hosts the optical

system, which, as previously stated, focuses the light incoming from the laser

through the first fiber, and deliveries the collected backscattered light to the second

fiber, which drives it directly to the spectrograph entrance slit. The two optical

paths, which join inside the optical system, are separated by a dichroic mirror, which

reflects on a perpendicular direction the electromagnetic radiation whose

wavelength is exactly 785 nm and is crossed by the higher spectral components. In

this way, the incoming laser beam is deflected towards the optical system, while the

returning light, backscattered by the sample, is separated in frequency. The elastic

component is reflected, while the Stokes component of the Raman scattering is

57

transmitted towards a notch filter (who further rejects the remaining laser line) and

coupled to the second fiber.

To our knowledge, this is the first Raman spectrometer which exploits a pair of

galvanometric mirrors to perform a dynamic map of a remote surface under

investigation; it is worth to notice that, even if the instrument described and built by

Puppels et al [II.28] adopts a similar scanning system, that is actually a Raman

imaging device which takes advantage of the periodic movement of the

galvanometric mirrors to produce an homogeneous illumination of the field of view.

Figure II.1. The layout of Remote Raman scanner. From left to right, the computer control, the

detection unit and the laser, lodged on a trolley, and the remote probe, positioned on a tripod in front

of the tripod.

II.5. RRS: the standard components

Semiconductor laser.

The adopted laser source is the Lion model, produced by Sacher Lasertechnik GmbH

(Germany). It is a small (165×60×54 mm3), light (0.6 kg) semiconductor laser,

working in continuous wave (CW) regime (figure II.2) and equipped with a Littman

external cavity: this configuration exploits a diffraction grating as one of the cavity

folding mirror. This element, illuminated by the laser emission through a collimating

lens, provides a spatial dispersion of the emission modes of oscillation of the laser

cavity, so that only the radiation of the chosen wavelength can reach the lasing

threshold. Mode hopping and frequency drift, typically related to semiconductor

58

lasers, are therefore suppressed: additionally, the high quality of the cavity

elements, together with a special anti-reflection coating of the exit surface, grant a

spectral width of only 500 kHz and a maximum output power of 270 mW [II.55].

The laser head is driven by a remote control (Pilot) which manages both the current

supply and the temperature control of the device: it is important, for the stability of

the light emission and the durability of the laser diode, that the temporal behavior of

these two parameters is smoothly varying and a continuous check of the status of

the light source is given as a feedback to the Pilot.

Fig. II.2. Layout of the Littman cavity laser. The active medium (in pink) is crossed by the laser beam

focused by two lenses (L1 and L2). The first lens collects only the emitted light which bounces back

from the mirror M, according to the selection performed by the diffraction grating G. Thanks to this

element, only radiation at very narrow wavelength can reach the laser threshold and leave the cavity

through lens L2.

The wavelength for the excitation of the Raman effect lies at the lower border of the

Near Infrared (NIR) range. This choice has been made in order to limit the possible

luminescence emission by the target material, which in many cases overwhelms the

much less effective Raman signal. Most of the luminescent transitions are, indeed,

triggered by radiation in the Ultraviolet (UV) or visible wavelength range. There are,

however, a number of drawbacks related to the adoption of a NIR laser source. First

of all, the quantum efficiency of a traditional silicon sensor decreases for

wavelengths above 700 nm; moreover, the non-resonant Raman cross section was

shown to be inversely proportional to the fourth power of the wavelength. However,

an excitation wavelength in the UV, which provides the maximum efficiency, does

not match the commonly available optical components, and therefore would have a

59

negative influence on the overall budget for the instrument. Additionally, the

spectral resolution of a Raman spectrum, ceteribus paribus, increases with the

wavelength; furthermore, a laser emitting in the UV or in the visible range results in

higher absorption rates by the observed samples, which enhances the risk of

photochemical or thermal degradation. For this reason, moreover, a CW laser has

been chosen, instead than a pulsed source. Even if the Lion® can provide quite a high

intensity, it should be noted that this extra power is needed to compensate the

losses introduced along the beam path by the optical fibers and the set of lenses. As

will be stated in the next chapters, though, the power density which was actually

delivered on the sample has always been kept within a safety limit (< 400 W/cm2).

Galvanometric mirrors.

The Remote Raman Scanner employs a VM1000+ (Cambridge Technology, USA)

galvanometric system for the deflection of the laser beam. The device is made of a

pair of mirrors, mounted on two orthogonal axes; each mirror is free to rotate,

within a permitted range, around its axis and its angular orientation is selected by a

current value. This system exploits, indeed, the alignment of a coil in which current

is made flow, with a permanent magnet, driven by the current value; the response of

the mirror, which depends by its moment of inertia, is generally very rapid (down to

0.5 milliseconds) and pretty accurate (the galvanometer can be used as a device to

measure currents down to 1 nanoAmpère). The considered system is designed for

many laser scanning purposes (marking and prototyping above all), and therefore is

not specifically conceived to work in a Raman spectrometer: some of its features,

such as the dimensions of the rotating mirrors (respectively 10 and 15 mm along the

rotating axis for the X and Y deflector), can be limiting in the development of the

planned optical path; nonetheless, the system is sufficiently small not to interfere

with the compact design of the overall probe head. Other features which make this

device compatible with the purpose of this project are its good angular resolution

(2×104 steps per each angle), the angular excursion of 80° along both the horizontal

and vertical axis, and the reflective coating of the mirrors for the NIR range.

60

Holographic notch filter.

The holographic notch filter selectively rejects (through Bragg diffraction) light

falling within a narrow spectral interval, while leaving unaltered the other spectral

components of the electromagnetic radiation which crosses it. It is constructed in a

photosensitive medium, dichromated gelatin, by exposing it to interfering laser

beams, which create a periodic modulation in the refractive index. This periodicity

produces a strong 3d Bragg reflection that can efficiently (> 99.9 %) diffract away

the Rayleigh line, while transmitting adjacent wavelengths with > 90 % efficiency

[II.56].

Being much more economical, practical and smaller than a dedicated

monochromator, the notch filter is the only possible choice to introduce a rejection

element for the elastically scattered light in a portable instrument, with a single

excitation line. Among the commercially available at the time of building the

instrument, the Iridian® ZX000419 notch filter provided the narrowest

transmission curve (with a Full Width Half Maximum of 22 nm) and a considerable

rejection of the elastic component, with an optical density of 4; with a diameter of 1

inch, this element was easily incorporated into the Remote Raman Scanner.

Czerny-Turner spectrograph.

For the dispersion of the spectral component of the Raman signal, the Remote

Raman scanner exploits the SpectraPro2150 (Acton Research Corporation, USA), a

Czerny-Turner (CT) spectrograph: actually, this is the most widely used

configuration for Raman spectroscopy. It makes use of mirrors as collimators in an

off-axis configuration and employs a planar reflective grating in the collimated

space. This design leads to a basic one-dimensional spectral dispersion dimension.

The entering light, in the original design, passes through a vertical slit, whose width

can be regulated by a precision knob.

The principal advantages of the CT configuration are its relative simplicity and

wavelength flexibility. Because it is an all-reflective design, it is naturally achromatic.

The CT is easy to scan and can accommodate multiple gratings, making it an

attractive spectrograph for research systems that require flexibility to operate with

many different lasers. Disadvantages of this design are in the areas of the etendue,

61

imaging performance, durability and thermal/mechanical stability. Vignetting at the

edges of the spectrum is relatively large due to the large distance between the

output collimator and the reflection grating. Thermal instability of the CT

spectrograph is about a factor of 5 higher than that of a similarly configured axial

transmissive spectrograph [II.56].

In the Remote Raman Scanner, SpectraPro2150 represents a trade-off between

performance and limited size, being its internal focal length 150 mm with external

dimensions of 178×178×165 mm. Its weight is just 4.5 kg, which is considerably

helpful for limiting the overall load of the instrument. The gratings mounted on the

single turret are two: they have respectively 600 and 1200 grooves per millimeter,

with a blaze at 750 nm, and can be rotated or interchanged via a stepper rotary

encoder. The first one grants a wider spectral windows, while the latter, which is

used as default, allows for an higher resolution: with the narrowest slit width (10

µm), the discriminating power can be better than 0.4 nm. When employed with the

portable probe head, however, the performance are well below the resolution limits,

due to the intrinsic width given by the diameter of the fiber core, which can be 100

or 200 µm according to the target material’s Raman efficiency: in this configuration,

in fact, the entering slit is represented by the fiber tip, which is plugged to the

spectrograph through a FC port which exactly replaces the original slit.

Charge coupled device (CCD).

Charge-coupled devices (CCD) appeared as reliable, commercial products for

spectroscopic applications in the early 1990s: now, they represent the most

common technology for the detection of light in this kind of experiments. In

particular, this sensor meets more of the desired detector characteristic for Raman

spectroscopy than any other currently available technology. These characteristics

include the ability to measure many wavelengths at once, large dynamic range, low

read noise, low dark current, robust packaging and long detector lifetime. The CCD is

fabricated on MOS (metal-oxide semiconductor) technology. The charge is stored in

the capacitor gate and readout occurs after sequential charge transfers by

periodically controlling the voltages on the electrodes [II.56].

In the ultimate configuration of the spectrometer, a iDus (Andor, Northern Ireland)

front-illuminated CCD is used for the monitoring and acquisition of the collected

62

radiation: it is based on a rectangular silicon sensor of 1024×127 square pixels,

each one with a 26 µm width. The device is equipped with a pre-amplifier and a 16-

bit analog to digital converter and a double-stage Peltier cooling circuit which

permits a refrigeration of the chip down to -70°C in standard conditions. Its

dimensions are 157×100×87 mm3. Whereas a back-illuminated sensor (in which

the chip is flipped upside down and the silicon-active medium is reduced in

thickness such that light enters the silicon from the back side) can be chosen to

enhance the quantum efficiency (QE), the disadvantage of using these types of

detectors for 785-nm-excited Raman is detector etaloning [II.56]. This phenomenon

can be prevented by working in the deep depletion regime, but the dark current

noise is considerably enhanced. For these reasons, and for a considerably lower

price, a standard front-illuminated sensor has been selected. Dynamic range is

generally expressed in terms of the number of bits produced by its digitizer.

However, a significant parameter is the full well capacity of each pixel,

correspondent to the maximum amount of stored charged in each CMOS: in this

particular camera, this value is of 106. The number of the elemental charges is then

read by the digital converter with the possible application of a gain factor; the full

well capacity, however, allows a direct comparison with the typical noise figures of

the sensor, which are also expressed in electrons. In this case, the readout noise is

estimated as 3 electrons, while the dark current is typically of the order of 2×10-3

electrons per pixel per second. under normal conditions, these noise figures are too

low to hamper the detection of the Raman signal [II.55], which is much more

threatened by the competition with luminescence background.

Optical fibers.

Optical fibers provide to the system layout the needed flexibility in order to design a

compact probe head and, at the same time, to maintain a bulkier but high-quality

part for the acquisition and elaboration of the Raman signal. It would be impossible,

indeed, to arrange all the necessary instrumentation on a single transportable

working surface: therefore, some parts of the optical path need to be guided by a

flexible medium. If there is no particular issue for the delivery of the laser beam to

the probe, because the losses can be easily compensated by an higher exit power of

the laser source, introducing losses to the already weak signal is much more

63

disadvantageous. For this reason, a multiple choice for the second fiber, from the

device probe to the spectrograph, is allowed: the main option is a FC-connected,

100 µm core, fused silica, 3-meters long graded-index multimode fiber, equivalent to

the one used from the laser source to the optical system. Its numerical aperture is

0.29. This choice allows a discrete coupling with the backscattered radiation, while

offering a smaller diameter which results in good resolution spectra. If, however, the

collected spectra does not show a sufficient signal to noise ratio, or the exposure

time to achieve an unambiguous spectrum is too long with respect to the

experimental demands, a second option is available: a FC-connected, 200 µm core, 3-

meters long step-index multimode fiber provides a more efficient collection of the

backscattered light, with the drawback of hampering the resolution of the dispersion

element. Examples of spectra collected with both the configurations will be shown in

the next pages. The multimode fibers allow the connectors and other optical

elements in the path, such as the laser injector, to be less critical in alignment [II.57].

In its last configuration, RRS takes advantage of the coupling by optical fibers to

switch to a laboratory version of the instrument which adopts a more traditional

layout. This transformation is achieved by connecting the laser source and the

detection apparatus to a fixed Raman probe, equipped with a interchangeable

microscope objective and mounted on a micrometric translator along the three

Cartesian axes. The converted instrument is adopted when, for laboratory analysis, a

more precise single-point analysis is needed: in this way, with an easy operation of

disconnecting and plugging two optical fibers, a second set-up is obtained.

II.6 RRS: the custom-made systems.

The optical system.

As shown in Fig. II.1, the design of the optical system stems from the requirement of

directing the parallel laser beam deflected by the 2-axis galvanometric scanner to

any point on the surface of interest. Hence, the X, Y coordinates in the area of

interest are mapped by the θ, ϕ angular positions of the galvanometric (galvo)

mirrors. Reversing the optical path, the Raman light collected by the optical system

64

is delivered to the galvo mirrors in a collimated beam and deflected towards the

detection unit. Since this task is definitely more critical than the handling of the laser

light, the analysis of the optical system described henceforth refers to the gathering

of the Raman scattering. Three main requirements have been critical in the design of

the optical system. The working distance of the Remote Raman Scanner should not

be shorter than 20 cm; this is the minimum value that grants a practical in situ

employment of the instrument. A safe non-contact working distance is preferable, as

previously mentioned, for the analysis of works of art. The second requirement

concerns the need for satisfactory mapping of heterogeneous paintings or

sculptures, and therefore the development of an instrument capable of analyzing

areas on the scale of tens of square centimeters. A flat field and absence of vignetting

over the whole field of view are also needed to achieve semi-quantitative Raman

measurements. A further issue that has challenged the design of the optical system is

the very low cross section of Raman scattering. This results in the need for as large a

numerical aperture as possible in order to maximize the collection of Raman signal.

To fulfill this requirement, a frontal lens with a 51-mm diameter has been chosen.

Considering a working distance for the optical system close to 20 cm, this leads to an

f-number (f/#) around 4. On the other hand, to limit the weight of the head of the

Raman spectrometer, the diameter of the scanning mirrors has been limited to about

10 mm. This means that the optical system must be able to direct as much Raman

light as possible from a large entrance pupil (51 mm) to a smaller exit pupil. A final

critical point is represented by aberrations: while chromatic aberration is virtually

negligible due to the narrow band of the Raman signal, spherical aberrations limit

the field of view of the optical system. After an in-depth study of the light path, a 4-

lens system was chosen as the simplest option for the construction of the device.

Specifically, a sequence of a positive lens, a negative lens, and finally two positive

lenses provided the desired properties of the light path. The adoption of a higher

number of lenses would provide a higher flexibility, but at the same time it would

increase the aberrations and the losses due to reflections. When considering a 4-lens

system, the values of the 4 focal lengths and those of the 3 distances between the

lenses are the parameters of the project, yielding a total of 7 degrees of freedom. In

addition, lens diameters and glass types could be used as additional parameters, but,

for the sake of simplicity, we decided to work with off the shelf (commercially

65

available) lenses, all having the same diameter of 51 mm. This choice reduces

assembling complexity and project costs.

After a preliminary project based on standard lens design criteria, the optimal

configuration of this system was optimized using the ray tracing software ZEMAX

(Development Corporation, USA). A virtual replica of the scanning device was

implemented for simulating the optical path. For the sake of simplicity, the ray

tracing was simulated only in a 2D space: this is a reasonable approximation, since

the optical system shows cylindrical symmetry. In addition, Zemax allows the

comparison between lenses of different manufacturers: the very same design has

been tested using lenses of seven brands. The simulations were addressed to

maximize a figure of merit that describes the Raman scattering power that crosses

the exit pupil of the optical system for each detection point on the surface of interest.

According to the simulations, Optosigma (USA) manufactures the elements that best

fit our specifications: the choice or this project was to adopt an aspheric lens of 50

mm focal length f.l. , followed by a divergent lens f.l. mm and a pair of

identical biconvex lenses (f.l. = 52.7 mm). The optimal distances between these

lenses were 20, 31, and 45 mm: the distance between the last lens and the galvo

mirrors is then determined by the position of the exit pupil, and is equal to 35 mm.

66

Fig. II.2. The quoted project of the optical system; distances between the lenses have been optimized

by Zemax.

Fig. II.3. Simulated collection efficiency ε r for the optical system as a function of the displacement of

the sample along the plane of the field of view, for a surface at a distance of 20 cm from the external

lens

67

The final project configuration is shown in Fig. II.2. If the theoretical beam path was

drawn, it would be possible to see that not all the collected light reaches the

scanning mirrors. In order to theoretically evaluate the capabilities of the designed

optical system, we simulated its collection efficiency ε r as a function of the radial

position on the surface of interest. ε r was estimated as the percentage of the

Raman light, scattered by the point on the sample surface at distance r from the

optical axis, which is able to cross the exit pupil. A Lambertian distribution was

assumed for the Raman radiant intensity. The result is shown in Fig. II.3: it is

possible to observe that the theoretical field of view is ∼12 cm in lateral size, and

that the collection efficiency is reasonably uniform within this area. It should be

further noted that the simulated collection efficiency is rather low (close to 0.3%).

Nevertheless, this value is close to the theoretical collection efficiency of an ideal

optical system with an f/# of 4 (approximately 1.5%). The lower efficiency of the

designed optical system is mainly ascribable to the smaller size of the footprint of

the scanning mirrors compared to the exit pupil. A better performance could be

achieved by increasing the mirror size, within the limits of loading allowance of the

galvo scanner; bigger deflection set-ups are available on the market, but they are not

tailored to this kind of employment and therefore they would represent a bulky and

unconfortable alternative for the overall probe dimensions and weight [II.54].

The software.

An user-friendly virtual interface was developed in order to manage all the devices

which can be controlled by the computer, and at the same time to monitor, acquire

and organize the collected data. The aim of the software, called RRS, is to provide to

the researcher an high-level access to the experimental parameters which he or she

reasonably needs to interact with, while hiding the most basic technical instruction

which must be given to the machines.

The software was programmed in the C++ language, and the code has been written

and compiled with the help of the LabWindows® suite (National Instrument, USA).

The control of the several devices relies on the libraries furnished by the producers

of the CCD, spectrograph and galvanometric scanner (Andor, Acton Research and

Cambridge Technology respectively).

68

In its current version (2.0), it is possible to manage the galvanometric mirrors, the

spectrograph and the CCD, while the parameters belonging to the laser source

(emission wavelength, exit power) can be inserted manually: in the next release, the

laser will be controlled by software, as well. It is planned, as well, to connect a

webcam to the probe head, and to show the image of the target area in a inset of the

interface window.

Taking inspiration from the most common commercial software, the program menu

offers a variety of options, which range from three operating procedures to the

instrumental settings to the usual file managing commands. What is unique in RRS

with respect to similar applications, is the possibility to set different points of

analysis and watch the system automatically deflecting the laser focus on the

selected position and starting the measurement. The possible working regimes are

three: the Run mode is used to monitor the evolution of the signal in real time,

without any average operation; Acquire is used for a standard measurement where

the laser spot position is fixed; Scan, instead, performs the analysis on the set of

selected points, acquiring and showing a spectrum per each position of the focus.

The file menu, in the end, allows the loading and the saving operations for one

spectrum or a set of spectra.

The graphic interface of RRS is shown in picture II.4: the main commands which can

be given to the instrument are distributed according to the system they refer to. The

selection of the laser focus position inside the field of view and the definition of the

pattern of the various analysis positions are some of the available operations in the

Mirrors sub-menu. The Spectrograph bar, instead, allows the user to choose the

diffraction grating and to position it to a specific central wavelength position. The

CCD menu is the most complex. A window for the temperature control of the

charged coupled device opens automatically when the software is started, and can

be accessed at any moment between the acquisitions, to set the cooling options. The

Region of Interest (ROI) of the silicon chip in which the analysis must be performed

can be defined via a dedicated window, as well as the vertical binning, if the sensor

is acquiring in imaging mode. The default operations, however, occur in

spectroscopy mode: this option enables a full vertical binning of the pixels, so that

the main window shows the intensity profile of the captured light, which, if properly

calibrated along the horizontal axis, corresponds to the spectrum. With regard to

69

this aspect of the measurement, a preliminary operation to each working session is

the acquisition or the loading of a reference spectrum, which ensures the correct

correspondence between the horizontal pixels and the chosen spectral coordinate

(frequency, wavelength, wavenumber, relative wavenumber): this task can be easily

accomplished via a dedicated panel. The last important parameters to be submitted

to the program are the exposure times for the acquisitions. Again, three possibilities

are given to the user, according to the working regime. The researcher can decide to

set a fixed exposure time, which will be applied to the single or to all the points of

analysis; otherwise, different exposure times, set by the user, can be applied to each

position of the laser spot; finally, an automatic procedure can be started, which

regulates the exposure time in order to achieve the selected signal intensity. This

functionality employs a trial-and-error method, in which the software guesses the

correct exposure time, tests it and then evaluates if the chosen value satisfied or not

the required signal to noise ratio: in this way, RRS automatically moves towards an

optimal exposure time; however, to prevent an exceedingly long process in

particularly unfortunate cases, a maximum number of repetitions can be entered. It

is worth to stress that, beside the exposure time of the single acquisition, a number

of accumulations can be planned per each spectrum: this method performs a

summation of the collected spectra of the same point of analysis to reduce the

statistically random contribution of the shot noise to the signal.

RRS software exhibits a communication window in its upper part, to provide the

user confirm of each inserted parameter, and to report the successful or not

execution of the given commands. The set of experimental parameters, furthermore,

are saved in the header of each spectrum file (bearing the e tension “.rrs” and can

thus be recovered together with the acquired data.

70

Fig. II.4. the graphic interface of RRS software in a typical Raman measurement. The yellow led on the

left top of the window indicates that a Scan measurement is running, and the coloured bar in the

lower part of the interface shows the progress of the acquisition.

71

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77

CHAPTER III.

RRS AT WORK: TESTS AND CASE STUDIES.

The previous chapter introduced the Remote Raman Scanner by explaining the

driving concept behind its development. This chapter reports the several tests which

have been performed to evaluate its behavior with respect to the planned

characteristic: the instrument performances have been assessed through

experiments with known samples. After a positive validation, the device has been

used for a series of analysis, on a heterogeneous set of samples, both as a punctual

source of information and as a mapping technique. Unfortunately, no chance to

employ the device in situ occurred until now; yet, the set-up was adopted for the

reported measurements in its ultimate configuration, and therefore ready to be

transferred without altering its structure; moreover, the long working distance

proved to be a useful property also for laboratory analysis.

III.1 Characterization of the performances.

Ad hoc tests have been performed to assess the depth and the flatness of the field of

view, the spatial resolution and the spectral accuracy of the developed system. A

series of other tests have been carried out on standard samples of several pigments

and binding media in order to appreciate the effectiveness of the device with

different materials and to evaluate the typical signal to noise ratio which can be

achieved for this kind of analysis; the spectra, not provided here, were collected in a

little database, in order to be used as references for comparison.

78

Assessment of the depth of the field of view.

The first test aims to the determination of the dependence of the collected Raman

signal on the displacement of the target object along the optical axis (z). For the

purpose, a Cinnabar (HgS) reference was placed in front of the Remote Raman

Scanner and moved along the z axis within the distance range 18.5-24 cm with a step

size of half centimeter. A Raman spectrum was collected per each position, with an

acquisition time of 10 s and a power density on the sample of 300 W/cm2;

successively, the intensity of the main Raman band of HgS, correspondent to Hg-S

symmetric stretching, (νs = 252 cm-1 [III.1]) was plotted with respect to the z-axis

position. The resulting curve is shown in fig. III.1. It is possible to notice how the

system provides a smooth variation in the collection efficiency in the selected range:

an estimation of the depth of field is given by the full-width half maximum (FWHM)

of the curve, approximately equal to 60 mm. The maximum efficiency occurs at a

working distance of 20.5 cm, a value which is in good agreement with the project

specification for the focal length.

Fig. III.1. Intensity curve of the 252 cm-1 Raman band of HgS, collected at different positions of the

target sample along the optical axis.

79

Assessment of the field of view.

The dependence of the collection efficiency on the focal plane has been evaluated as

well. A ColorChecker White Balance Card (X-rite, USA) is a board 20.9 x 27.9 cm2

wide covered with a uniform Titanium white (TiO2) layer, typically used for

checking the light uniformity of the field of view [III.2]; here it was employed as a

spatially uniform sample, and placed at the optimal distance from the most external

lens of the Raman scanning device (20.5 cm). A double line-scan was performed on

the two perpendicular axes (x and y) which cross the optical axis. The whole range

permitted by the mirrors deflection (± 4 cm in both directions) has been explored,

with a step size of 0.5 cm; the employed laser power density on the sample was of

300 w/cm2, while the acquisition time was of 20 s per each point. The intensity of

the reference Raman band of Titanium white, corresponding to the Ti-O symmetric

stretching (442 cm-1) was extracted from each spectrum, and after baseline

correction, it was recorded. The plot of the two curves obtained along the x and y

axes can be seen in figure III.2. Despite being both curves slightly asymmetrical, it is

possible to appreciate that within the interval -30 mm, +25 mm along both axes,

choosing the optical axis as the origin of the frame of reference) the collected

intensity is fairly flat. An estimate of the width of the possibly analyzed area, by

considering again the FWHM, is about 65 mm for both directions: for higher angular

deflections, the collection efficiency decreases quite drastically.

This area is smaller by a factor of 1.5 with respect to the simulated one. This

overestimation is mainly due to the fact that the virtual replica of the galvo mirrors

can freely rotate, while the VM1000+ scanning mirrors have a limited angular range

(±40°); the laser beam, therefore, can be deflected within a solid angle which is

contained within the aperture of the optical system, but that does not fill it

completely.

80

Fig. II.2. Intensity profile of the 442 cm-1 Raman band of TiO2 as collected from different points of the

target surface perpendicular the optical axis, along the horizontal (x) and vertical (y) coordinates.

Assessment of the spectral accuracy.

The spectra obtained by the latter measurements were also exploited to evaluate the

spectral accuracy of the Remote Raman Scanner. In other words, the shift on the

wavenumber axis undergone by the vibrational band at 442 cm-1, taken as a

reference, was measured for each focus position. This shift is actually an artifact due

to slightly different coupling of the backscattering light with the optical fiber which

“feeds” the spectrograph: its highest value was equal to cm-1, a result which is of

the same order of magnitude of the spectral resolution of the instrument, and much

lower than the FWHM of the TiO2 Raman band (19 cm-1). It is hence evident that the

uncertainty of the spectral coordinate in the Raman spectrum is negligible with

respect to the resolution of the spectrograph.

Assessment of the spatial resolution.

For the applications of purpose, the Remote Raman scanning does not need

extremely high spatial resolution: variations of the vibrational signal on micrometric

distances are more suitably revealed by a Raman microscope. Theoretically

81

however, the granularity of the galvo mirrors allows a precision of the order of 4 µm

(80mm of lateral width/ 20000 angular steps) in the focus positioning. On the

contrary, the laser spot size is approximately 200 µm wide because of the particular

choice on the employed optical system, and in order to maintain a low risk of

damaging the target sample with an high power density. However, it is necessary to

provide at least an indication of the spatial resolution, in order to estimate the

uncertainty at which the position of the analyzed point can be determined.

The spatial resolution of the scanning device was derived by the measurement of the

Edge Response Function (ERF). This is the intensity curve obtained when observing

an ideal step profile and it is strictly related to the spatial resolution of the device. In

fact, this quantity can be estimated through the so-called “rise distance” of the RF,

i.e., the distance for the signal intensity to change from 10% to 90% in

correspondence of the edge. The same definition can be adopted to indicate the

reversed distance (90%–10%) in the falling edge.

In order to assess this feature, a reference sample was created by making two

grooves with a width of 800 μm, separated by a distance of 10.5 mm, on a flat

aluminum surface. Each groove was filled with Titanium white (powdered Rutile).

The spatial transition from metal (no signal) to the synthetic mineral (Raman

signal) can be considered as an ideal edge. The sample was scanned along a line

perpendicular to the two white stripes with a variable step size equal to 70 μm in

correspondence of the edges and to 300 μm in the region between the stripes. It

should be noted that the intensity profile (fig. ZZ) of the two grooves is slightly

asymmetric, since the falling edges are steeper than the rising one: this must be

assigned to a non-perfect alignment of the sample with the field of view.

Nonetheless, if the average value is taken of the the “upwards” and “downwards”

edges, the result does not lose its significance.

The mean “rise distance” as defined above of the intensity of the Raman signal of

TiO2 by considering both of the two white stripes was estimated as 430 ± 130 μm

(Fig. 6). It can be inferred that the spot size of the focused laser beam does not

represent a limiting factor for the resolution of the device.

82

Fig. III.2. Intensity profile of the 443 cm-1 Raman band of TiO2 contained in two 800 µm-wide grooves

(whose position is shown in gray) on a metal surface.

III.2 A multi-analytical approach to a model painting.

The set-up has been used to record spectral information from a model panel

painting naturally aged in ambient conditions for 25 years. The model was prepared

by prof. Aviva Burnstock of Courtald Institute of Art, London, using traditional

pigments from a local supplier including lead white, gypsum, ultramarine (a sodium

aluminium silicate), and vermillion (HgS) bound in an egg-based binding medium

(Fig. III.4) [III.3]. The panel is divided into two smaller paintings, both of which have

been interested by the different studies. The left one is characterized by an area

covered by a gilding; the latter will not be covered by Raman spectroscopy

measurements. Two other techniques, among the most common for this kind of

studies, have been employed: reflectance multispectral imaging and X-ray

Fluorescence.

The necessity to use more than one analytical technique for a single identification

and diagnosis set of measurements is indeed well known [III.4]. For this reason,

several mobile instruments are employed together at the measurement site [III.5,

83

III.6] or, alternatively, multipurpose devices are being developed [III.7]. The purpose

of this laboratory test is to reproduce, in little, a in situ analysis of a work of art, and,

in particular, to validate the results supplied by the Remote Raman spectrometer

with data coming from complementary methods.

Fig. III.4. Model panel used to test the ability of the Remote Raman Scanner to identify pigments. The

materials and techniques employed for its realization are faithful to traditional egg-based painting.

Reflectance spectra and reconstructed maps.

The reflectance multispectral (rMI) imaging unit is a portable instrument which

acquires a series of reflected images of the object of interest, each one in a narrow

spectral interval. The device consists of two voltage-stabilized 150 W tungsten

halogen lamps (DLH4, Dedotec, USA) placed approximately at an incident angle of

45° with respect to the normal of the painted surface. The radiation reflected from

the surface is acquired with a low noise CCD camera (Retiga2000, QImaging,

84

Canada) coupled to a band-pass liquid crystal tunable filter (Varispec, CRi, USA),

which is used to scan across the visible spectrum. The measurement protocol

involves the acquisition of 65 images, each one related to the light reflected by the

surface in a different spectral band from 400 to 720 nm in 5 nm steps. The non-

uniform illumination of the field of view is corrected with the aid of a white balance

paper (Gretag Macbeth ColorChecker White Balance, USA); for the estimation of the

reflectance factor ρx,y( ) in each point of the field of view, data calibration is

performed using a calibrated 99% diffuse reflectance standard (SphereOptics, USA)

[III.2].

Each measurement provides, thus, a set of 65 images (1200×1600 pixels); if the

intensity values of a single pixel in each image are collected in a 65-elements array,

the reflectance spectrum of the point corresponding to that pixel is obtained. This

means that the result of each measurement is multi-spectral cube, a 3d matrix

whose dimensions are the two spatial coordinates corresponding to the points of the

image, and the spectral coordinate which varies in the interval 400-720 nm.

The raw data cube, therefore, provides straightforward information when it is

sectioned by a plane parallel to one of its faces. However, in most cases a more

synthetic expression of the collected data is needed in order to proceed with a more

refined analysis. For example, areas of the painted surfaces characterized by

different spectral features are highlighted by applying a supervised classification

algorithm based on the similarity between a reference reflectance spectrum (chosen

in correspondence to a certain area in the field of view) and each reflectance

spectrum of the analyzed surface [III.8]. The approach based on the Spectral Angle

Mapper classification (SAM) has been followed, where both the reference spectrum

and the measured spectrum ρx,y( ) at location (x,y) are treated as vectors. The

spectral angle between them, calculated according to the following equation,

, arccos ,

,

is taken into account as the similarity index, small angle values suggesting a high

degree of similarity.

(III.1)

85

For the panel under investigation, four reference spectra, corresponding to the most

common distinguishable colors, have been selected (Fig. III.5). The first one,

indicated as data1, corresponds to a point in the dark blue shadow of the lady’s vest,

in the right picture; the second refers to the red color adopted for the semicircle

surrounded by the guilding, this in the left-hand part of the panel. The third selection

(data3) has been made on a pink area corresponding to the lady’s cheek; the fourth,

instead, is located on the pale skin of the lady’s face.

Fig. III.5. Reflectance spectra of the sampled points on the panel. Data1refers to the blue color in the

painting, data2 to the red, data3 to the pink and data4 to the white.

Similarity maps were reconstructed for each of the selected spectra: in figure III.6

they are listed according to the chosen order. Each pixel is colored on a gray scale,

according to the value of the spectral angle between the reference spectrum and the

spectrum of that point: white indicates a null angle and therefore perfect similarity,

black the opposite.

86

Fig. III.6. Similarity maps of the painted panel, obtained with the selected reference spectra. From top

to bottom, left to right: similarity map for the blue (data1), red (data2), pink (data3) and white

(data4) color respectively.

The similarity maps conveys an immediate description of the distribution of the area

with analogous reflectance spectrum; this representation provides a much more

powerful information than a single reflectance map, revealing the spatial

distribution not of a single spectral band, but of a set of spectra. In the case of

interest, the panel painting results clearly segmented into four images which can

almost be superimposed without overlaying. Even though in this case study the

selected colors are significantly different, the adopted method is able to reveal

spectral similarities also in less distinguishable situations [III.2].

Despite the fact that reflectance imaging alone cannot, unless very favorable cases

[III.9], reveal the identity of a pigment, it is clear how it can significantly simplify the

role of subsequent analytical methods: in this case, the identification of the pigments

can be limited to four points of the studied area, without losing in the generality of

the collected information.

87

XRF analysis.

X-rays are electromagnetic waves emitted during highly energetic electronic

transitions within the atoms provoked by the bombardment of a target with charged

particles (electrons or ions) or with high energy radiation. They can be used for

obtaining transmission images of the works of art (radiography) or as a

spectroscopy source. In the second case, a detector is used to collect the

fluorescence photons emitted after X-ray interacting with the target material. Since

this high-energy fluorescence is related to the atomic energy structure, X-Ray

fluorescence (XRF) is a fast spectroscopic technique to provide the identification

and, possibly, quantification of the atoms contained into the object of interest. Being

the X-ray penetration depth inversely proportional to the atomic mass, almost all the

elements having an atomic number above a certain threshold can be detected. In

portable detectors, especially useful for the analysis of cultural heritage, the lighter

detectable atom is usually sodium 11Na. The limit to detectable elements is given by

the presence of air and by the entrance window of the detector, which is usually

made of a low atomic number element (Beryllium).

In collaboration of XGLab, a spin-off company of Politecnico di Milano, a series of

Energy dispersed X-ray fluorescence (ED-XRF) punctual measurements have been

performed on the painted panel. For the analysis, the latest portable device, called

ELIO, was employed. Equipped with a large area Silicon drift detector (25 mm2), it

can work in high resolution or in fast mode according to the input photon energy. Its

excitation source is a transmission X-Ray generator consisting of an Rh anode; the

instrument is particularly suitable for the analysis of cultural heritage thanks to a

couple of pointing laser (one on the axial and one on the focal direction), and to a

microscope camera which monitors a 20×20 mm2 area around the analyzed point:

the working distance is of the order of few millimeters. With a weight of 2.1 kg, ELIO

is a truly portable instrument: for the sake of reproducibility and comfort, however,

it was not hand-held during the measurement, but lodged on a tripod.

Four points of analysis (plus one on the gilding) have been chosen on the panel

surface, according to the selection performed through reflectance imaging. The

measurements have been performed in acquisition times of about 40-60 s each, and

the emitting tube was supplied with a 40 kV voltage (20 µA of current). The

resulting spectra have been reported in figure III.7 the portable instrument

88

automatically provides an interpretation for the distinguished peaks.. The top one is

related to the blue areas: the main constituting element is sulfur, with a small

percentage of lead. The latter being related to lead white pigment (lead basic

carbonate, 2PbCO3·Pb(OH)2), the only responsible for the blue color should be

sulfur. Among the traditionally available pigments, only lazurite (a.k.a. Ultramarine

blue, Na8(Al6Si6O24)Sn, n = 2-3) contains sulfur: a safe identification, however, is not

possible on this sole evidence. Despite the fact that sulfur is also a common element

in many other pigments, in this case the high relative concentration of this element

(~97%) suggests that it cannot simply be present in the underlying layers, but it

must actually take place in the blue pigment on top. Fluorescence emission of the

red spot, again, reveals a two-elements composition. In this case, however, the

relative concentration is almost equal to unity for the first one, mercury, whereas

iron is limited to the 0.71%. Mercury sulfide (HgS), as previously stated, can be

found naturally as cinnabar: from antiquity this mineral has been used as a pigment

and it is called vermilion. The iron oxide Fe2O3 constitutes another historical (and

archaeological) red pigment, red ochre. Its presence could be considered because

the statistical confidence interval, associated with the measurement, is quite high:

however, the low statistical uncertainty related to Hg, and the bright red color of the

selected area support the identification of the red pigment with vermilion.

XRF spectrum of the lady’s cheek, selected as the reference area for the pink color,

reveals the presence of lead, mercury and iron. This suggests a reasonable synthesis

of the pink color as a mix of lead white, vermilion and possibly red ochre: the iron

component (3%) in the fluorescence spectra is, indeed, not negligible in this case,

and red ochre could be used to provide a more realistic color to the skin. This

suggestion is confirmed by the spectrum of the white selected area, corresponding

to the lady’s nose and forehead. In this case, only lead and iron can be traced:

however, the relative composition of lead is increased, coherently with the

hypothesis of a mix of lead white and red ochre. No evidence of vermilion is here

found, and this is in agreement with the different reflectance spectra obtained for

the cheeks and the rest of the face.

Two more analysis have been performed out of the four reference areas, and

involved the white background of the two figures and the gilding. The first is rather

uniform throughout the panel, and thus a single acquisition was carried out. It

89

needed quite a long exposure time (180 s) to obtain the spectrum reported in the

higher part of figure III.7bis. The reason is justified by the table of the composition:

the two detected elements are rather light, and they are sulfur (~53%) and calcium

(~47%). The presence of this two elements in grossly equal percentages, hints at

gypsum (Calcium sulfate, CaSO4) as the ground layer which was likely used by the

artist.

The gilding was not expected to be real gold, still XRF reveals a non-negligible

(>13%) composition of this metal, the rest being mainly iron. Another significant

result is the detection of calcium, which at approximately 11% of the total

composition is the third detected element: not visible to the naked eye, this calcium

must probably be assigned to the gypsum used as the adhesive to fix the gilding to

the panel, according to one of the traditional methods for these decorations.

Sample name Element Concentration (%) Estimated error (%)

Blue S

Pb

96.94

2.8

2.20

0.99

Red Hg

Fe

99.29

0.71

0.64

5.63

Pink Pb

Hg

Fe

90.57

6.38

3.05

0.74

2.13

3.08

White Pb

Fe

98.77

1.23

0.56

6.54

Gilding Fe

Au

Ca

As

74.08

13.26

10.79

0.72

0.49

1.63

3.91

4.36

Tab. III.1. Elements detected by XRF in the selected areas of the panel.

Fig. III.7 (next page). XRF spectra of the four selected areas, from top to bottom: blue, red, pink and

white.

90

91

Fig. III.7 bis. XRF spectra of the white background (top) and of the gilding layer (bottom).

Flexible Raman mapping.

With the comfort of the results of the first two techniques, selective Raman mapping

was used to obtain a molecular analysis of pigments in the different colors over the

surface. No need for a raster scanning was here recognized, so the laser beam has

been focused only on few positions within the field of view of the instrument. The

measurements were performed at a distance of 20 cm from the panel, with a laser

power density of 200 W/cm2 on the sample and an acquisition time of 50 s for each

analyzed point (20 for the red area). It is important to underline that during these

inspections the painting was not moved.

The first test allowed the identification of ultramarine as the blue pigment: the

spectrum (figure III.8a), indeed, reveals the 548 cm-1 band of the lazurite pigment,

due to the symmetric stretching of the S- ion [III.1]; the 1007 cm-1 band of gypsum is

also present, hinting that the hue of the color was dimmed with the white pigment,

92

or, more likely, that the blue color does not fully cover the underlying gypsum

preparation layer. The identification of this pigment, thus, could only be confirmed

by a molecular-sensitive technique such as Raman spectroscopy, because in this case

the information provided by XRF are not sufficient (the element composing

ultramarine blue, N, Al, Si, O and, to some extent, S, are indeed too light to be

effectively revealed by that technique). No fingerprints of lead white was detected in

this area, but the Raman cross-section of this Lead compound is not particularly big

at 785 nm and its presence is probably hidden by the background noise.

The spectrum of the red pigment is not provided here because it is not very different

from the one obtained by the pink area, reported in figure III.8b: indeed, the Raman

fingerprints of Vermilion are very intense (282, 343 cm-1). The difference between

the spectrum of the pure red and the pink selected points is the presence of the 1050

cm-1 band of lead white in the latter. The results are in good agreement with the XRF

analysis, even if it was not possible to distinguish any of the spectral fingerprints of

red ochre, which is probably under the limit of detection of our instrument.

The spectrum of the white part of the lady’s face fig. III.8c shows two close little

bands, the first one at 1007 cm-1 must be assigned to gypsum, whereas the second,

at 1050 cm-1, reveals the presence of lead white. Again there is no trace of iron oxide,

but this is coherent with the Raman spectrum of the pink areas.

The last spectrum of figure III.8 refers to the white background of the panel, and it

indeed reveals all the spectral fingerprints of CaSO4 in the selected spectral window:

420 cm-1 and 494 cm-1 are related to the molecule’s out of plane bending, whereas

620 cm-1 and 674 cm-1 to the in plane bending. The intense band at 1007 cm-1,

instead, is assigned to the symmetric stretching while 1132 cm-1 to the asymmestric

stretching of the sulfate ion in CaSO4 [III.10].

93

Fig. III.8. Raman spectra of three of the selected areas, and (at the bottom) the spectrum of the white

background.

94

III.3 Identification of white and fluorescent pigments.

In , a Fluorescence lifetime imaging FLIM analysis on the an Gogh’s

watercolor “Les bretonnes et le pardon de pont ven” e posed at the Galleria d’ rte

Moderna in Milan, revealed an uncommon luminescence arising from some of the

white-colored areas in the painting [III.11]. Our research group is currently trying to

determine the substance which is responsible for this behavior: before a more

extensive study can be performed directly on the artwork, a series of original early-

XX-century white pigments and contemporary luminescent pigments have been

gathered and analyzed with different techniques. Some of the data which have been

supplied by FLIM and multispectral fluorescence analysis strongly suggest

Lithopone (∼60% ZnS and ∼40% BaSO4) is the pigment used by Van Gogh for the, of

course unintentional, luminescent areas. Another hypothesis, though, is that an

unusually doped zinc oxide (ZnO) has been used as the white pigment. Normally,

these two pigments do not exhibit a long-lasting fluorescence like the one present on

the watercolor, still the presence of an accidental doping agent within the

compound, caused by a non-refined synthesis, may have reasonably caused the

pigment to be luminescent.

Along with other non-destructive methods, thus, Raman spectroscopy was used to

characterize samples in powder of Lithopone “Chamot” reference collection

pigments. In figure III.9, the Raman spectrum of the pigment produced by ACME in

1922 is compared to the spectrum of a standard Barium sulfate powder. It is

possible to identify the original pigment as Lithopone because of the small band

arising at 340 cm-1, characteristic of ZnS and not present in the spectrum of BaSO4.

The distinction of this vibrational fingerprint, therefore, would be the main target of

a Raman study of the actual watercolor: still, not all the selected Lithopone pigments

provided a sufficiently resolved spectrum to detect it clearly.

95

Fig. III.9. Raman spectrum of the sample of XX-century Lithopone (black) vs the Raman spectrum of

the Barium white standard (gray). The exposure time was 30 s, 5 accumulations are used, laser

power density is 200 W/cm2.

In order to have a reference on the currently available luminescent pigments, two

other samples included the commercially available Fluoreszenz Grün, a white glow-

in-the-dark pigment, and Nach-Leucht-Pigment Grün, a greenish white pigment,

bought by Kremer® Pigmente GmbH (Germany). These materials have been used to

characterize the fluorescence produced by expressly luminescent materials,

obtained by highly-doped semiconductors, with possible emission resulting by

casual doping of traditional pigments. No identification of the substances within the

commercial pigments was supplied by the producing company. The Raman analysis,

however, was able to recognize at least the most important compound within each

pigment. Fluoreszenz Grün, indeed, apparently contains Zn2SiO4, since the bands at

390, 890, 904 and 944 cm-1 can be easily detected [III.12]. Pigment Grün, instead,

showed Raman bands at 218, 348, 416, 608, 634 and 667 cm-1 which can be

assigned to zinc sulfide (ZnS) [III.12]. ZnS, indeed, is the most used phosphor in

cathode screens, but its luminescence property is activated by doping with acceptor

materials, like copper. Being one of the two compounds present in Lithopone, zinc

sulfide is likely to be the responsible of the luminescent behavior of the an Gogh’s

96

watercolor. The presence of a doping agent, however, cannot be easily revealed by

Raman spectroscopy: a X-Ray Fluorescence analysis would be, in this case, more

significant.

Fig. III.10. Raman spectrum of the sample of Zinc Sulfate (black) versus the spectrum of Pigment

Gruen. No baseline subtraction has been applied to the spectra, whose intensities have been

normalized.

Fig. III.11. Raman spectrum of the sample of Fluoreszenz Gruen compared with the of standard Zinc

Silicate. The spectra have been normalized but not baseline subtracted.

97

It is worth to stress that none of the reported spectra undergone baseline

subtraction; despite being strongly luminescent, they did not exhibit a significant

background when excited with the 785 nm laser.

III.4 Analysis of samples from the “Memoriale italiano” of

Auschwitz.

In 1980, the Italian National Association of the Former Deportee (ANED),

inaugurated a memorial inside the 21 block of the concentration camp in Auschwitz.

The monument is made of a painted spiral which winds throughout the barrack

guiding the visitor inside the building (fig. III.12); Pupino Samonà, inspired by a text

of Primo Levi, realized the 23 stripes which compose the spiral. The object of

interest is an aerograph painting on textiles and it is under the supervision of

Accademia di Brera. Considering a restoration, the personnel of the Accademia is

currently collecting samples to be examined with different spectroscopy methods,

such as Infrared and Raman spectroscopy. Some of these samples, catalogued with

an abbreviation citing the number of the object and the stripe of provenience, have

been studied by the Remote Raman Scanner, thanks to a collaboration with

Emanuela Nolfo, a PhD student of the Accademia.

Some of the collected spectra are reported here. P6A1 is a white fragment of the first

stripe, and indeed it contains spectral fingerprints of white pigments, namely

titanium white (TiO2, in its rutile form) and calcium carbonate (CaCO3). The

reported spectra was collected in .. seconds with the 600 grooves/mm grating. P4A2

refers to a Red fragment, in which, however, the bands of TiO2(248, 442, 610 cm-1)

and CaCO3 (711 e 1088 cm-1) are dominant over any evidence of a red pigment or

dye. P5E23 refers to a yellow fragment: the grating has been rotated in order to

extract the spectral information from another window. The spectrum allowed the

identification with the arylide monoazo-dye based pigment Yellow 1, a.k.a Hansa

yellow [III.13]. Its recognizable bands are 997, 1137, 1215, 1254, 1310, 1390, 1455,

1487, 1532, 1567, 1599, 1622, 1670 cm-1. Thanks to the partial superposition of this

and the former spectral range, the 1087 cm-1 of Calcium carbonate can be detected

in this spectrum as well.

98

Fig. III.12. a. Plant of the Italian memorial of Auschwitz, hosted in one of the original barracks

of the prison camp. b. A picture of the of the painted spirals in the third room.

a

b

99

Fig. III.13. Raman spectra of some of the samples e tracted by Samonà’s work in the Italian memorial

of Auschwitz. Exposure time: 50 s. Laser density power: 100 W/cm2. No correction applied.

100

Subsequent analysis with a Fourier-Transform Infrared absorption (FT-IR)

spectrometer revealed the presence of a vinyl material within each sample, evidence

of the glue used as a binding medium; on a particular sample. P6A1, it was possible

to distinguish the spectral fingerprints of the fiber. They matched quite well the IR

absorption bands of cellulose, which reveal that the stripes have been made of

cotton. The presence of this organic material, together with the vinyl glue, is

probably responsible for the significant background present in the reported spectra,

which does not prevent, however, the identification of the most significant Raman

bands.

III.5 Study of a 3D plastic object

Polymeric materials have been used extensively in the production of design objects

thanks to the versatility of their mechanical, physical and optical properties, which

have been finely tuned through the addition of various plasticizers, stabilizers,

colorants, and blends of different polymers: the heterogeneity and sensitivity of

polymeric materials make the conservation and analyses of design objects

particularly challenging [III.14].

In this application, for the first time the Remote Raman Scanner has been adopted to

study a non-flat surface. The object of interests are polymeric design products of the

1950s-1960s; among them, it is worth to mention one of Castiglioni’s lamps,

Taraxacum. The reported spectra, though refer to the well-known Grillo telephone,

designed in 1965 by Marco Zanuso and Richard Sapper and manufactured in Italy by

Siemens. Being revolutionary in its shape and in its practicality, the phone can be

seen in museums of modern art such as the Centre Pompidou in Paris, or the MoMA

in New York City. The sample described here is part of the collection of the Triennale

Exhibition in Milan: it has been studied as part of the PhD thesis of Francesca Toja

on the conservation of polymeric materials [III.14]. The Grillo here concerned is

excellently preserved, though at a fluorescence multispectral analysis it provided

quite a different response for the microphone and the rest of the handset: fig. III.14b

101

synthetically reports the difference in the reconstructed RGB coordinates which

correspond to a slight variation in the maximum of the fluorescence spectrum.

The Raman analysis was performed, then, to try to ascribe this difference in the

fluorescence properties to a possible modification of the composing polymer, due to

a variation in the light exposure of the two sides. The Grillo phone, thus, has been

placed in front of the instrument and opened so that both the two areas could be

reached by the laser focus without moving the sample. The two spectra, together

with the reference spectrum [III.15] of the constituting polymer (Acrylonite

Butadiene Styrene – ABS), are provided in figure III.15.

As it can be noticed, no apparent evidence of photo-induced degradation of the

surface is provided by the Raman spectra. Probably, the variation occurred only to

the very e ternal layer of the handset, or the instrument’s resolution does not allow

a sufficient sensitivity to determine if the detected bands underwent a shift.

However, it should be stressed that the instrument did not fail in the identification of

the plastic compound, with a remote acquisition of 20s exposure time and 180

W/cm2 laser density power in order not to damage the telephone with an excessive

dose of photons.

Fig. III.14. A picture of the Grillo in its open and close configuration, on the left. The reconstructed

RGB fluorescence image of the upper, lower and lateral views of the telephone, on the right.

102

Fig. III.15. Raman spectra of the two selected areas of Grillo, compared to a reference spectrum of

ABS

III.6. Mapping of a rock sample.

The capability of working with non-flat surfaces makes the Remote Raman Scanner

particularly advantageous also in situations that are not strictly related to the

Cultural Heritage framework. Thanks to a collaboration with the geologist Sabrina

Moneta of the Statale University of Milan, a number of rock samples have been

studied: the case reported here is chosen to show once again the capability of the

instrument to perform a flexible Raman scan of the target surface. In this case,

however, the collected set of data has been processed by another self-made

software, in order to produce Raman spectral maps of the considered area. Called

RamanViewer, this program has been developed with Matlab® R2007B and it

103

provides a comfortable interface to manage the data acquired and saved by RRS.

Among its other features, what is relevant to this purpose is the possibility to

reconstruct spectral map of the scanned area for a single or for an interval of

vibrational shifts. The user can specify the starting and ending vibrational position

of a selected Raman band (for instance, 1077-1097 cm-1 for the 1077 cm-1 band of

Calcium carbonate) and the program plots the intensity of the selected spectral

interval for each analyzed area, taking into account its spatial coordinates. The

resulting image is virtually insensitive of the underlying background, since the

software exploits an algorithm that calculates the area comprised between the

spectrum and the line connecting the two extremes specified by the user. Thanks to

this calculation, a preliminary normalization of the spectra is not needed to obtain a

meaningful spectral map.

The selected rock, mainly made of argillite with vains of calcite mineral, is pictured

in figure III.16: the analysis covered an area of approximately 20×15 mm2 across

one of the calcite vains. The measurements were performed at a distance of 20 cm

with a laser power density of 500 W/cm2. Exposure time was set as a constant for all

the 24 points on which the laser has been focused, and it was equal to 40 s; no

baseline subtraction was applied to the spectra. Figure III.17 reports three spectral

maps which, despite the very thin sampling of the selected points, clearly permit to

distinguish the spatial profile of three vains. Indeed, they have been obtained by

reconstructing the intensity map of the spectral intervals 270-292 cm-1, 1075-

1100 cm-1 and 455-480 cm-1 respectively: the first and the latter being consistent

with the most notable spectral feature of CaCO3 (282 and 1087 cm-1,), whereas the

second refers to the SiO2 symmetric stretching at 469 cm-1. The reported spectrum

represents the point of coordinates (-4.52, 0.96): no baseline subtraction was

applied. For clarity, also the band of CaCO3 at 710 cm-1, not used for the map, has

been marked.

This application, while not particularly interesting from the point of view of the

analysed samples, is aimed once again to evaluate the mapping performance of the

Remote Raman scanner on a particularly irregular shape. Within a collaboration

project with the Grand Egyptian Museum of Gyza, a series of tests on fragments of an

Egyptian cartonnage and of a wall of an Egyptian city dating to the Roman period

(31 b. C., 313 A.D.), have started recently with promising results, since the coverage

104

of the cartonnage and the decorations of the wall are mainly made of inorganic

materials. The next step is, then, the Raman mapping of this two samples: the second

will probably provide the first occasion to test the Remote Raman Scanner in situ.

Fig. III.16. Photograph of the argillite + calcite sample. The red square shows the central area of the

RRS field of view. The red dots correspond to the positions from which the spectra have been

collected.

105

Fig. III.17. a. Spectrum of the area of coordinates -4.52,0.96 of the surface. b. Maps of the area under

the bands at 289, 470 and 1087 cm-1 respectively. It is worth to notice that, while belonging to the

spectrum of Calcium carbonate, the spatial distribution of the first and the last chosen band is not

completely overlapping; the spatial distribution of SiO2 (470 cm-1), instead, is concentrated in the

second row on the right.

a

b

106

Bibliography.

[III.1] D. Marano, I. M. Catalano, and A. Monno, Pigment Identification on “Pietà” of

Barletta, Example of Renaissance Apulian Sculpture: a Raman Microscopy Study,

Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 64 (2006),

1147–50;

[III.2] D. Comelli, A. Nevin, G. Valentini, I.Osticioli, E. Castellucci, L. Toniolo and

others, Insights into Masolino’s Wall Paintings in Castiglione Olona: dvanced

Reflectance and Fluorescence Imaging Analysis, Journal of Cultural Heritage, 12

(2011), 11–18;

[III.3] A. Brambilla et al., A remote Raman scanning spectrometer for in situ

measurements of works of art, RSI (2011);

[III.4] C. Fotakis, D. Anglos and V. Zafiropoulos, Lasers in the preservation of Cultural

Heritage: Principles and Applications, Taylor & Francis (2006);

[III.5] C. Miliani, F. Rosi, B. G. Brunetti and A. Sgamellotti, In Situ Noninvasive Study

of Artworks: The MOLAB Multitechnique Approach., Accounts of chemical research,

43 (2010), 728–38;

[III.6] T.D. Chaplin, R. J.H. Clark and M.Martinón-Torres, A Combined Raman

Microscopy, XRF and SEM–EDX Study of Three Valuable Objects – A Large Painted

Leather Screen and Two Illuminated Title Pages in 17th Century Books of

Ordinances of the Worshipful Company of Barbers, London, Journal of Molecular

Structure, 976 (2010), 350–359;

[III.7] I. Osticioli, N.F.C. Mendes, A. Nevin, A. Zoppi, C. Lofrumento, M. Becucci, E.M.

Castelluci, A New Compact Instrument for Raman, Laser-induced Breakdown, and

Laser-induced Fluorescence Spectroscopy of Works of Art and Their Constituent

Materials, Review of scientific instruments, 80 (2009), 076109

[III.8] A. Pelagotti, A. Del Mastio, A. De Rosa, A. Piva, Multispectral Imaging of

Paintings, IEEE signal processing magazine (2008) 27–36.

107

[III.9] M. Bacci, M. Picollo, G. Trumpy, M. Tsukada, D. Kunzelman, Non-invasive

identification of white pigments on 20-th century oil paintings by using fiber optic

reflectance spectroscopy, The American Institute for Conservation of Historic &

Artistic Works, 46 (2011), 27–37;

[III.10] .D. Chaplin, R. J.H. Clark, D. Jacobs, K. Jensen, and G. D Smith, The Gutenberg

Bibles: Analysis of the Illuminations and Inks Using Raman Spectroscopy, Analytical

chemistry, 77 (2005), 3611–22;

[III.11] D. Comelli, A. Nevin, A. Brambilla, I. Osticioli, G. Valentini, L. Toniolo, and

others, ‘n the iscovery of an Unusual Luminescent Pigment in an Gogh’s Painting

“Les Bretonnes t Le Pardon e Pont ven, Applied Physics A, (2011);

[III.12] RRUFF, database of Raman spectra online (www.rruff.info);

[III.13] N. Scherrer, S. Zumbuehl, F. Delavy, A. Fritsch, and R. Kuehnen, Synthetic

organic pigments of the th and st century relevant to artist’s paints: Raman

spectra reference collection. Spectrochimica acta. Part A, Molecular and

biomolecular spectroscopy 73, no. 3 (August 2009): 505-24;

[III.14] F. Toja, A. Nevin, D. Comelli, M. Levi, R. Cubeddu and L. Toniolo, Fluorescence

and Fourier Transform Infrared Spectroscopy for the analysis of iconic Italian design

lamps made of polymeric materials, Francesca Toja. Analytical & Bioanalytical

Chemistry, 399 (2011) 9, 2977-2986;

[III.15] Tsuchida, Akihiro, Hirofumi Kawazumi, Arikata Kazuyoshi, and Tuchida

Yasuo, ‘Identification of Shredded Plastics in Milliseconds Using Raman

Spectroscopy for Recycling’, 2009 IEEE Sensors, (2009), 1473–1476;

108

CHAPTER IV

SERS ANALYSIS OF AMINO ACIDS WITH A

PORTABLE INSTRUMENT.

A different application of a portable Raman instrument is here described. This

chapter is more concerned on the employed technique, which is Surface-Enhanced

Raman Spectroscopy, than on the instrumentation: the described experiments,

indeed, involve the adoption of a commercial portable instrument for the SER

analysis of liquid and solid samples of amino acids. No in situ (with the meaning of

“out of laboratory” S RS e periment is, to date, published: this work, despite

relying on laboratory tests, validates the effectiveness of SER analysis of simple

molecules performed with a portable spectrometer. The purpose of this study is to

determine the crucial parameters and the critical points of this kind of application,

in order to pave the way for a real on-site employment of this method on samples of

cultural or archaeological importance.

The work here reported is part of a collaboration program between Politecnico di

Milano and the FOundation for Research and Tecnology – Hellas (FORTH) in

Heraklion. The present text reports the results of a research period of five months at

the Institute of Electronic Structure and Laser (IESL-FORTH) of Heraklion, under the

supervision of professor Demetrios Anglos.

IV.1. The aim of the work.

As introduced before, this research is a contribution to a wider project which has the

objective the development of new portable instruments and techniques for the

analysis of organic residuals in archaeological finds. The identification of these

remains, often extensively affected by age and degradation, is possible through the

109

detection of organic molecules (e.g. amino acids, fatty acids) which act as bio-

markers specifically related to particular compounds. There are a number of

situations, however, like the screening of the presence of organic substances, the

growth of fungi or the incidence of contaminants on a valuable object or monument,

where chemical analysis must be performed directly on site, and almost immediately

[IV.1]. The use of this type of in situ analysis, which can provide meaningful results

in a reasonable time, is particularly critical for archaeologists, conservators and

scientist involved in such studies. In this work, simple organic molecules were

chosen to assess the performance of SERS analysis with a portable instrument.

Amino acids, the building blocks of proteins, are common analytes for SERS and

several approaches for enhancing the Raman signal of these molecules have been

developed: past research, though, exploited bench-top Raman microscopes. Here, a

portable Raman spectrometer is applied to liquid and dried samples of amino acids

and small peptides in aqueous solution; the contribution of silver or gold colloid to

recovery the Raman fingerprints, lost after reducing the concentration of the

analytes, is evaluated.

The purpose of this study is not the development of a highly specific detection tool

for one molecule, but the evaluation of the minimum requirements for the device

and for sample preparation in order to obtain diagnostically significant spectra. In

this perspective, no ad hoc procedure was exploited to maximize the Raman cross

section of a specific sample. Although some considerations about particular cases

are reported, this work should be considered within a framework of the study of

biological samples and within the context of archaeological residue analysis.

IV.2 SERS of amino acids.

Amino acids are organic molecules in which one amino group (-NH2) and one

carboxylic acid group (-COOH) are connected to a so-called side chain, another

group specific for each amino acid. These compounds are the building blocks of

peptides, and hence, of proteins: their presence is, therefore, widespread in all

samples of biological origin. For this reason, they have been among the first

molecules to be analyzed with Raman spectroscopy [IV.2]; however, only samples in

crystalline form can be revealed without significant effort, and therefore their

110

spectra can be a valid reference only to a certain extent. G. Zhu et al. showed that

Raman analysis of 18 amino acids in various concentrations (but always above

0.01 M) is nowadays possible with a bench-top micro-Raman device, but most of the

spectral fingerprints are lost in the passage from solid to solution (especially for the

aromatic amino acids) or are altered due to shifts and broadening of the bands

[IV.3].

In general, several analytical techniques are employed for the detection and analysis

of proteins: according to the applications, chemical decomposition techniques can be

employed (hydrolysis followed by separated by ion-exchange chromatography or

hydrophobic interaction chromatography), or mass spectrometry. In archeological

applications, however, reference must be addressed to other techniques which,

while being sensitive for these molecules, are most suitable for the analysis of

residues or traces of materials contained in ancient vessels or fragments of pottery.

Normally the identification of these remains relies on different laboratory

techniques, including Fourier-Transform Infrared spectroscopy (FTIR) and Gas

Chromatography-Mass Spectrometry (GC-MS) [IV.4, IV.5].

As a diagnostic tool, Surface Enhanced Raman Spectroscopy (SERS) is particularly

suited for the analysis of organic molecules present even in very low concentrations

[IV.6,IV.7]. Many studies on proteins and amino acids have been carried out,

revealing the presence of these analytes in solutions (as low as 10-6 M [IV.6]). The

need for micro-samples, together with the requirement for minimal sample

preparation are the drawbacks of typical SERS measurements, yet they are largely

compensated by the massive increase in the signal to noise ratio and hence

sensitivity provided by the use of a suitable nano-structured substrate. In addition

to signal enhancement, SERS has a fluorescence quenching effect, which is extremely

desirable for the analysis of biological samples, as those usually exhibit a high

fluorescence background in Raman spectra excited by visible laser radiation [IV.8].

Experiments exploiting different materials, such as silver colloids, synthesized

following several recipes, gold containing chromatographic beads, silver films or

silver-coated glass fibers via Tollen’s reaction are described in the literature. In

particular, however, most of the reported methods focus on the use of metal

electrodes whose surface is artificially roughened before they are inserted in a

suitable cell containing the molecules in solution [IV.9, IV.10]; Stewart and

111

Fredericks, however, used a silver electrode as a substrate but left amino acid

solutions to evaporate [IV.11]. In the analysis of amino acids as part of this work,

silver and gold colloids synthesized according to Lee and Meisel’s and Leopold and

Lendl’s methods are used, not only because of their simple and cheaper production

coupled to their very good performance with a wide range of laser wavelengths and

analyte molecules: the main reason behind this choice is to test the portable

instrument with one of the most practical substrates if a in situ operation is

foreseen: silver and gold nanoparticles, indeed, if stored in a dark and refrigerated

environment, do not need any particular care before being applied and can be used

without any significant efficiency loss up to one month after their synthesis [IV.7].

SER analysis of amino acids, in synthesis, can be a key passage in developing an

effective non-destructive diagnostic technique for archaeological remains. However,

its role in the Cultural heritage field would not be limited to this application, thanks

to its high sensitivity to the molecules and their behavior, even in small

concentrations. The mechanisms involved in the chemistry of painting can be

interpreted, for example, with time-resolved spectral SER analysis: for example, this

method allowed Cañamares et al., [IV.12] to monitor the binding process between

alizarin and ovalbumin, employed in egg tempera.

The promising results obtained in the application of SERS must be consistent with

those furnished by other techniques, and above all, face particular problems

regarding experimental reproducibility: it is known, indeed, that spectral distortions

can occur with SER spectra. A key problem for the analytical application of SERS, and

especially for the characterization of biological samples, is to attain reproducible

signals combined with a large enhancement factor. The reproducibility of SERS

spectra and the achieved enhancement depends on many experimental factors,

including nature and morphology of SERS substrates, geometry of the analyte-metal

interface, as well as the excitation wavelength [IV.6]. This work will try to evaluate,

as far as possible, which of these factors influences the measurement most.

Nonetheless, a more complete set of experiments, using a bench-top

instrumentation, is necessary for a thorough description of the SERS of amino acids

which is beyond the scopeof the current project. In order to test the portable Raman

device as a suitable instrument for in situ SERS, research was focused on the

applicability of the method to samples of increasing complexity.

112

IV.3 SERS with metal nanoparticles.

A wide variety of SER substrates has been developed and is currently under test.

Among the first to be introduced are gold and silver nanoparticles, and they are still

one of the favorite choices for many analyses. The main reason is that their synthesis

does not rely on particularly expensive or sophisticated techniques or apparatus,

but they can be obtained via a chemical process that exploits the reduction of salts

or acids containing these two elements. The most reliable and most well-known

methods for synthesis were published in the 1982 by Lee and Meisel [IV.13]; the

majority of SERS measurements is based on these substrates [IV.14- IV.17]. It is

convenient, indeed, to exploit the silver colloid as a preliminary test for SER

measurements on a wide variety of substrates, and then to resort to more specific

nano-structured materials in case the obtained sensitivity is not sufficient. On the

other hand, in spite of relatively simple preparation methods, the production of

reproducible colloids with the desired size and shape seems to be difficult, hence

limiting the reproducibility of the resulting SERS spectra [IV.6].

113

Fig. IV.1. a TEM image (a) of the silver colloids, and their absorption spectrum (b).

This method, or metal nanoparticles in general, has found several applications in the

analysis of Cultural heritage: it has been applied for the detection of proteins

revealing binding media, of inks and organic dyes. All these substances, indeed, give

rise to a significant fluorescence background which hampers the detection of the

Raman spectra. In most cases, the analysis using SERS has been carried out on

replicas of painted layers or inked paper; when the analysis of the actual object of

interest was needed, the measurements have been performed on microscopic

samples, removed by surgical blades [IV.18], or extracted by superimposing a

polymer bead loaded with a delicate acidic solution [IV.19] on a sample. However, in

one interesting case, it was possible to perform SERS directly on the object of

interest, a 16th-century Turkish carpet [IV.7]: in this case, metal colloid was the only

114

practical possibility to enhance the Raman signal of a selected area (a bunch of

textile fibers) without altering the target object or the instrument set-up.

In the current project, one of the silver colloids was prepared according to two of

Lee and Meisel’s procedures [I . 3]. Briefly, an aqueous solution of silver nitrate 8

mg of AgNO3 – Sigma Aldrich, USA) in 0.1 L of ultra-pure water (Millipore) was

heated to its boiling point and 2 mL of trisodium citrate aqueous solution

(Na3C6H5O7, 1% – Sigma Aldrich, USA) was added; the solution was kept boiling for

one hour: macroscopically, this colloid (Ag-LM) was characterized by a warm

grayish color. A second solution (Ag-HA) was obtained starting again from silver

nitrate (18 mg/l) in 0.1 L of ultra-pure water; 4.5 ml of NaOH (0.1 M) was mixed to

5 ml of Hydroxylamine –Hydrochloride (NH2OH ·HCl, 0.06 M) and then was added

dropwise to the aqueous solution of silver. The mixture was then stirred at room

temperature until milky grey color is obtained. This method has been introduced in

2003 by Leopold and Lendl [IV.20] and can be more effective for laser excitation in

the NIR. gold colloid u was prepared following again Lee and Meisel’s method

involving trisodium citrate as a reducing agent. The starting compound for the

reaction is chloroauric acid (HAuCl4): a solution 10-3 M is obtained by mixing 240 mg

of HAuCl4 with 0.1 L of ultra-pure water and brought to boiling. Then 50 ml of a 1%

v/v sodium citrate is added and the resulting solution is kept boiling for 1 hour. All

the solutions have been conserved in a dark and refrigerated environment (4 °C)

and they were always stirred before application. They have been used for one month

after the synthesis and in this period they demonstrated a good stability.

The formation of the nanoparticles was followed by monitoring the surface Plasmon

resonance band on a Perkin Elmer Lambda 950 UV-Vis absorption spectrometer

from 200-1000 nm: additionally, Transmission Electron Microscopy (TEM) was

carried out with a JEOL JEM-2100 Electron Microscope operated at 200kV, equipped

with a high resolution digital imaging CCD camera (model 782, ES500W

ErlangshenTM Gatan Inc, USA) to take images of the nanoparticles. In figure IV.1a it

is possible to observe the TEM image of a sample of the Ag-LM colloid, dried on a

glass microscope slide. The observed nanoparticles are roughly spherical, and their

diameter is of the order of 50 nm. In figure IV.1b, the absorbance spectrum is

reported: the Surface Plasmon Resonance is located at 431 nm, in good agreement

with the published spectra of the colloid [IV.13, IV.21].

115

IV.4 The portable instrument.

Analysis of solid amino-acids using Raman spectroscopy outside of the laboratory

environment has been reported recently by A. Culka et al. [IV.22]. In this work a

similar approach is taken, with the difference that well-established SER substrates,

i.e. silver and gold colloids are adopted in an effort to exploit a higher detection

sensitivity [IV.1].

A mobile Raman Spectrometer (HE 785, Horiba – Jobin Yvon, France) is employed:

as shown in Figure IV.2, it is based on a 785 nm semiconductor laser source coupled

by an optical fiber (ø = 50 microns, 5 meters long) to a compact probe head which

contains the necessary optics for sample excitation and signal collection. The

focusing lens can be chosen from a set of microscope objectives with different

magnifications: for the experiments considered here, a 20x (0.2 NA) and a 50x (0.4

NA) objectives were used. Collection of the Raman scattering signal is provided by

the same objective which delivers the backscattered light through an edge filter (OD

= 4, cutoff = 53 cm-1) to a second optical fiber (ø = 50 µm, 5 m long) which is

connected to a concave fixed grating (950 grooves/mm) spectrograph and a Peltier-

cooled CCD detector (Horiba Synapse). This setup performs measurements in the

range 50-3200 cm-1, with a spectral resolution of 4 cm-1. Light power on the sample

can be regulated from a maximum of 30 mW to a minimum of 0.3 mW. The

LabSpec® software package was used for spectra acquisition and data handling. A

VGA camera is seated inside the mobile probe head to monitor the area under the

field of view of the objective lens: when a mirror is inserted via a dedicated lever in

the light path, the instrument can be operated as an ordinary microscope. The whole

head (20×15×30 cm3, 8 kg) is mounted on a precision micrometer stage, able to

move it along X, Y, Z directions: together with the viewing camera, this allows

correct positioning and focusing of the probe. The size of the whole set-up is such

that it can be mounted on a mobile support (60×40×80 cm3 approximately,

considering the control computer).

This system was described by Westlake et al. [IV.23] and it has been recently used at

the Archaeological Museum of Heraklion for the analysis of pigments on a wall

painting. The same system, equipped with another laser source and normally used

116

for the analysis of ceramics, glasses and enamels in museum collections, already

demonstrated to be suitable for in situ analysis: micro-Raman spectroscopy, indeed,

was performed on rock art in South Africa [IV.24] with a HE532 micro-Raman

spectrometer by Horiba Scientific (Jobin – Yvon, France) under very uncomfortable

working conditions.

Fig. IV.2. The layout of HE785. The interior of the probe head is shown in the grey box, whereas the

laser source and the detection unit (light blue) are connected with optical fibers. The movable mirror

(M) which intercepts the laser beam (red) to enable the vision by the viewing camera is shown in its

two possible configurations; EF (standing for Edge filter) discriminate between the inelastic

scattering and the reflected component of the collected light.

IV.5 Tests and results.

The considered amino acids are L-Arginine (L-Arg, (NH2)2-C-NH-(CH2)3-CH-(COO-

NH2+)), L-Methionine (L-Met, CH3–S–CH2–CH2–CH–(COO-NH2+)) and L-

Phenylalanine (L-Phe, (C6H5–CH2–CH–(COO- NH2)+)); a more complex molecule was

represented by one tri-peptide, Glutathione (γ -L-glutamyl-L-cysteinylglycine). They

117

serve as model compounds referring to building blocks of proteinaceous materials

found in archaeological residues. Considering the evaluation of the proposed SERS

methodology and of the mobile instrument used, the choice of amino acids was

based on their structures. One of the amino acids has an aromatic side chain (L-Phe),

one contains sulfur (L-Met) while L-Arg has only a nitrogen rich side chain. The

selection of these amino acids was made for two complementary reasons: to explore

any favorable sensitivity arising from side chain chemistry and at the same time to

avoid possible over-estimation of the SERS effect (for example, the bonding between

sulfur and the silver nanoparticles), in agreement with criteria outlined by Sharma

et al. [IV.25].

The analyses reported below include:

- Raman spectra of the amino-acids in their crystalline form and in solutions

10-1 M;

- SER spectrum of L-Met in solution 10-3 M;

- SER spectra of the amino acid solutions dried on a layer of silver

nanoparticles;

- SER spectra of dried L-Met solutions starting with different pH values;

- SER spectrum of a dried mixture of L-Met and L-Phe;

- SER spectrum of a tri-peptide (Glutathione).

Raman spectra of amino acids.

The selected amino acids have been acquired from Sigma Aldrich (USA) in their

crystal phase. On these very samples a set of preliminary Raman measurements was

performed in order to determine the order of magnitude of the signal intensity

which can be revealed, in normal condition, with the portable apparatus. The

spectra of the three amino acids, L-Arg, L-Met and L-Phe are reported as figures IV.4

a, b and c respectively: in all cases, the objective of choice was the 20× and the light

power density on the sample was approximately 250 W/cm2. For all samples the

acquisition was performed in 10 accumulations of 20 s each.

118

Fig. IV.3. The molecular formula of the three amino acids of choice. From left to right: L-Arg, (NH2)2-

C-NH-(CH2)3-CH-(COO-NH2+); L-Met, CH3–S–CH2–CH2–CH–(COO-NH2

+), L-Phe, (C6H5–CH2–CH–(COO

NH2+-)

It is possible to notice a series of well defined bands for each spectrum: the

resolution supplied by the grating is not a limiting factor for the discrimination of

the spectral features. In general, most spectral features are all are contained in the

interval 300 – 1600 cm-1, but for L-Arginine and L-Methionine it is possible to

distinguish also the bands of CH2 stretching at 2920 cm-1 and, for L-Arg, CH3

stretching at 2870 cm-1. In the next paragraphs, however, attention will be focused

on the”fingerprint” spectral window -1800 cm-1, because the major detected

bands fall in this region..

The most significant detected Raman bands for the three amino acids with spectral

attributions are listed in tables IV.1, IV.2 and IV.3 for L-Arg, L-Met and L-Phe

respectively.

119

Fig. IV.4. Raman spectra of amino acids in powder. From top to botton: L-Arg, L-Met, L-Phe

120

In addition to Raman spectra of solid samples, spectra of the 10-1 M solutions of the

three amino acids have been collected; however, it was not possible to detect

significant spectral fingerprints with the reported instrument. Even exploiting a

highly sensitive laboratory Raman microscope (Nicolet Almega XR, Thermo

Scientific, USA), only L-Arginine and L-Methionine provided some recognizable band

structures. Despite the weakspectral features obtained by the portable instrument

HE785, an approximate quantitative assessment of the potential of the instrument

can be made. In figure IV.5 the signal obtained of L-Met in 50 s of acquisition at full

laser power is shown. The broad band arising between 1600 and 1700 cm-1, marked

by an asterisk, is due to the presence of water [IV.26]. Despite the very low quality of

the spectrum, it is possible to estimate the height of the Raman band at 1350 cm-1:

this intensity will be referred to the intensity of the correspondent SERS band to

obtain a rough estimate of the enhancement factor (EF) which can be obtained from

this measurement.

Fig. IV.5. Raman spectrum for 0.1 M L-Met. The asterisk marks the Raman band of liquid water

(symmetric stretching) at 1630 cm-1.

Analysis of liquid samples.

New solutions, obtained by gradual dilution of the 10-1 M of L-Arg, L-Met and L-Phe,

at intervals of 1:10 in volume, up to 10-6 M, were prepared. Three samples for each

of the three 10-3 M solutions were mixed in equal parts with Ag-LM, Ag-HA, Au

colloids respectively: in particular, a small volume (0.2 ml) of each mixture was

121

poured in a small, cylindrically shaped groove, machined on the surface of an

aluminum sample holder. Spectra have been collected 15 minutes after the solutions

were mixed; measurements have been performed by the 20× objective, the

maximum laser power (30 mW) on the sample and the average acquisition time was

50 s, with 10 accumulations per spectrum.

Since Ag-LM was the most effective colloid, every other sample solutions have been

mixed with this particular set of nanoparticles, with the same experimental

conditions (different parameters are explicitly reported). In general, however, the

quality of the SER spectra was not sufficient for the identification of the analyte. One

of the best results was obtained with L-Met (Fig. IV.6b). In contrast to normal Raman

spectra, the presence of Ag colloids in the amino acid solutions led to the

observation of several characteristic vibrational bands. It is possible to distinguish

several spectral features: 567 cm-1 (COO- wagging) [IV.22], 642 cm-1, 686 cm-1 (CS

symm. stretching), 770 cm-1 (CS stretching), 841 cm-1 (CC stretching), 925 cm-1, 950

cm-1 (CH3 rocking), 1031 cm-1 (CC stretching), 1181 cm-1 (CH stretching), 1363 cm-1

(CH3 symmetric stretching, COO- symmetric stretching) [IV.22], 1507 cm-1 (CN

asymm. stretching).

The Enhancement Factor (EF), defined in Maier [IV.27], with respect to a reference

solution prepared without Ag nanoparticles, cannot be calculated straightforwardly,

because of the absence of any visible Raman bands in the latter case. However, the

amplitude of the small band at 1350 cm-1 is observed in the Raman spectrum of the

10-1 M solution and was used to divide the height of the same band in the SER

spectrum: by taking into account the ratios between the concentrations and

between the exposure times, an indicative value for EF of approximately 2000 is

obtained. The spectrum is comparable to that reported by H. Lee et al. [6], if we

consider that the laser power is lower by a factor of 10 and the wavelength is further

from the nanoparticle plasmon resonance (785 vs 514 nm). With similar

considerations, the spectrum quality is similar to that acquired by Podstawka et al.

[IV.14] with a silver colloid synthesized by using NaBH4, even though they report a

larger number of bands.

The spectrum of 10-3 M L-Met was the most straightforward to acquire, with respect

to both the more and the less concentrated solutions of L-Met, which presented

distinguishable bands for concentrations as low as 10-5 M: it can be inferred that at

122

10-3 M the most effective coverage of the silver substrate is achieved for Methionine,

in agreement with Graff et al. [IV.9]. No unambiguous signal, however, could be

obtained from the L-Arg and L-Phe liquid samples. Therefore, further tests were

focused on dried samples of the same solutions: the concentration, from now on,

refers to the initial solution chosen as the sample, which though not strictly valid on

the solid sample, may be considered as indicative of the superficial density of the

adsorbed molecules.

Analysis of dried solutions.

Dried samples were also produced on a flat aluminum substrate, which does not

exhibit a significant background signal in the Raman spectrum. These samples are

not simply the dried version of the liquid mixtures, but a two-step procedure was

adopted to generate them. First a droplet (10 μl) of the silver colloid was placed on

the aluminum substrate and left to dry. The stain formed was covered with an equal

volume droplet of the amino-acid solution that was allowed to dry without heating.

This method is similar to the one described by Stewart et al. [IV.11] for the analysis

of amino acids on a roughened electrode; another comparable approach was

adopted by Huang et al. [IV.28], who heated the deposited colloid to remove the

solvent and aggregate the silver nanoparticles. Analysis was carried out with the

50× objective lens, and power density below 103 W/cm2 on the sample: integration

time, unless otherwise specified, was 10 s with 10 acquisitions per spectrum.

In figure IV.7, the SER spectra of L-Phe (10-4 M, a), L-Met (10-3 M, b) and L-Arg (10-4

M, c), deposited and dried over the evaporated colloid are shown; the main

detectable bands are labeled and assignments are reported in Tables 1, 2 and 3

respectively.

Following baseline subtraction the signal to noise ratio increases drastically with

respect to the liquid solution samples. This is particularly noticeable for spectra

acquired in pro imity of the “coffee ring” of the stain, where the density of the

deposited silver colloid is higher; the spectra are, nonetheless, representative of the

whole samples, and similar to typical spectral fingerprints found all over the surface.

The effectiveness of the metal layer produced by the evaporation of the colloid over

the substrate, together with the higher light collection provided by the 50× lens

with respect to the 20× on a relatively flat surface, combine to yield unambiguous

123

spectra for the three different amino acids, which are acquired in a time on the order

of one minute. Again, it is difficult to give an estimation of the EF because the Raman

spectrum of the same samples dried without the nanoparticle substrate does not

show any distinguishable features. Nonetheless, it is observed that the adopted

nanoparticles exhibit a preferential enhancement for bands in the spectral region

around 650, 1000 and 1300 cm-1. A comparison of the results obtained with L-Arg

and L-Met against other SER spectra recorded in similar works [IV.9, IV.29] indicates

that we detect a higher number of bands, whereas for L-Phe the quality of the

spectrum is similar to the one obtained by Lee et al. with silver nanoparticles and a

514.5 nm laser [IV.30].

Fig. IV.6. SER spectra of Methionine mixed with Silver colloid in liquid (b) and dried form (a). The

intensity scale of the b spectrum is multiplied by a factor of 40.

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Fig. IV.7 SER spectra of L-Phe (a), L-Met (b) and L-Arg (c) dried solutions (10s acquisition time, 10

scans, 104 mW/cm2 power density on the sample, 50x objective lens). The spectra are baseline

subtracted and normalised to maximum intensity for clarity.

Dependence of L-Met spectra on pH.

Amino acids in an aqueous solution may exist in three different forms, the anion, the

zwitterion or the cation, with the relative proportions determined by the isoelectric

point (pI) of the amino acid and the solution pH. The SER effect is known to be

strongly influenced by pH [IV.10, IV.28, IV.29]: with reference to the work of Graff et

al. [IV.9] and Abello et al. [IV.10], in this work the influence of pH variations on the

detection of SER signal was assessed only for L-Met. The pKa values of Methionine

are 3.3 for the carboxylic and 10.5 for the amino groups [IV.9], while the isoelectric

point is at 5.7: to monitor the evolution of the vibrational spectrum from the cationic

to the zwitterionic form, thus, a limited but significant range of pH values is

explored. The original solution of L-Met 10-3 M had a pH of 5.5, in agreement with

other references [IV.31]; two other 10-3 M solutions of Methionine in water were

prepared and adjusted to pH of 3.5 and 6.8 by adding HCl and NaOH respectively.

125

Since the quality of the spectra for the liquid samples does not allow many

considerations about the analytes in solution, it is necessary to follow the dry

approach also to monitor of the pH dependence. Dried samples obtained by the

standard 10-3 M L-Met solution, and two solutions at the same concentration with

adjusted pH values were analysed (with 20 s acquisition and 5 accumulations): the

results are shown in figure IV.8, where spectrum a refers to the cationic form (pH =

3.5) and c to the zwitterion (pH = 6.8). In both cases, the main distinguishable

bands, relative to C-S stretching for different rotamers, are located between 650 and

750 cm-1 [IV.31]: in this region some bands do not show significant shifts, but the

broad band at 662 (pH = 3.5) appears as a much narrower band and is shifted to

686 cm-1 when the zwitterion form is dominant. Another relevant spectral change is

the disappearance of the strong signal at 1037 cm-1 present in the cationic form

spectrum and related to C-N stretching. The CNH deformation, moreover, shifts from

1513 to 1542 cm-1 when the pH is raised. If the comparison is extended to the

spectrum b of L-Met at pH 5.5 (close to isoelectric point), however, identifying

simple tendencies in the alteration of the SER spectrum shape of many bands

becomes problematic; which is reflected in the range of shifts for Met reported by

others [IV.9, IV.10, IV.31]. The concurrence of possible conformers (TG, GT, TT and

GG [IV.31]) for L-Met produces a sequence of closely-spaced vibrational bands

which often overlap according to the shifts caused by the different environment

acidity. A thorough examination of multiple contributes was attempted by Lee et al.

[IV.32] by deconvolution of the broad bands through Fourier Transform analysis.

While such a deconvolution is beyond the scope of this work, it is important to note,

that the proposed method yields spectra with several of the characteristic bands for

this amino acid. The reported spectral fingerprints are, indeed, correspondent to the

ones mentioned by the cited authors for L-Met (Table 2).

Distinguishing two amino acids.

In addition to the simple amino acids solutions, mixtures were investigated. For

examples, L-Met and L-Phe solutions (10-3 M) were mixed in equal volumes before

being deposited with the usual method onto the dried silver colloid. This more

complex sample is produced in order to test a more challenging situation, in which

vibrational bands arising from different components of the mixture fall in

126

neighbouring frequencies. In Figure IV.9, a sufficiently well-defined spectrum of

both amino acids allows the assignment of vibrational bands associated with L-Met

and L-Phe, despite the broadening effect related to SERS: the bands at around 450

cm-1, as well as those at 528, 646, 681, 717, 960, 1091, 1130, 1506 and 1650 cm-1 of

L-Met are clearly distinguishable from those of L-Phe, and only some minor shifts

are observed in the mixture with respect to the SER spectrum of L-Met (Fig IV.7 b).

While the CC ring deformation band at 1004 cm-1 is recognizable in both Fig IV.7a

and Fig IV.9, other bands ascribed to L-Phe occurring at 859 (CC stretching), 1031

(CH stretching), 1207 (phenyl group stretching), 1275, 1357 (COO- stretching),

1418 cm-1 [IV.30, IV.33] are also observed in the L-Met:L-Phe mixture.

127

Fig. IV.8. Three spectra for methionine recorded at different pH values. From top to bottom, pH = 3.5,

pH = 5.3 and pH = 6.8. The upper spectra have been vertically shifted for clarity.

Fig. IV.9 SER spectrum of the mixture L-Met: L-Phe. 10-4 M after drying on the silver nanoparticles

layer. The vibrational bands of Methionine are written in bold, whereas the Phenylalanine spectral

fingerprints are written in italic.

128

Towards a more complex sample.

Fig. IV.10. Molecular structure of Glutathione (γ-L-glutamyl-L-cysteinylglycine)

Glutathione (γ-L-glutamyl-L-cysteinylglycine) provided an example of a naturally

occurring tri-peptide. Analysis of this larger molecule was performed on a droplet of

a 10-4 M solution, dried on the evaporated Ag-LM colloid. In this case the Raman

spectrum of the crystalline powder is provided (fig. 7) to show how the abundance

of bands in the Raman spectrum of the molecule in its solid form is substituted by

fewer, albeit wider, spectral features in the SER spectrum. This phenomenon can be

ascribed to the loss of order occurring when the Glutathione was dissolved in water

[IV.3]. Despite the significantly lower resolution of this SER spectrum, it is possible

to trace many of the vibrational modes of the molecules: 447, 622, 678, 760, 796,

925, 1050, 1281, 1326, 1367, 1634 cm-1, which are assigned in Table IV.4 [IV.28,

IV.34]. Again the most intense spectral features of Glutathione appear at around 650,

1000 and 1300 cm-1. A higher signal to noise ratio is obtained with respect to the

comparable work of Larsson and Lindgren, who used a laser in the NIR range but

gold colloid as the metal substrate [IV.34].

129

Fig. IV.11. SERS (a) and Raman (b) spectra for Glutathione. The spectrum a was recorded with 50x

objective, 5 s acquisitions (10 accumulations) and a laser power of 15 mW; spectrum b was obtained

with 10 accumulations, 20s each, laser power 7.5 mW and 20x obj.

IV.5 Perspectives.

The employed technique (SERS on dried mixtures) and instrument were

remarkably effective in the detection of simple molecules such as amino acids,

independently of the features of their side chains. The proposed method, however, is

intrinsically limited for the identification of more complex molecules or mixtures.

Even if many of the spectral fingerprints can be recovered after the addition of silver

colloids, the overall shape of the spectra is altered. First of all, sequences of closely

spaced narrow bands in the Raman spectrum of the molecules in crystalline form

become a single wide band when diluted. Even if the sample is dried after mixing

with the silver nanoparticles, the “fine structure” of the vibrational spectra is not

fully recovered, resulting in more ambiguous identification of specific spectral

features. This broadening of the Raman bands cannot be assigned to the resolution

of the instrument, which remains the same for the two measurements. It is worth

considering, in further analyses, the effect of the different kinds of synthesized

substrates with the dry approach, to investigate if gold colloids or Ag-HA

130

nanoparticles give a better spectral resolution, even if the enhancement factor, as

previously stated, is on average lower.

More generally, however, the main issue of this technique is the distortion of the

spectral profile which often affects the data obtained , even in the same sample.

These variations in intensity, which do not affect the spectral position of the main

vibrational bands, are caused by the strong dependence of the enhancement on the

local size of the nano-features of the surface [IV.35]. It is well-known that only those

molecular components that are sufficiently close to the SERS active surface (within

∼10 nm) and that have certain spatial orientation can be enhanced [IV.36]. Indeed,

the SER effect can occur if the resonance frequency of the excited SP matches that of

the scattered electric field [IV.37]. A more homogeneous colloid can be obtained by

controlled synthesis involving, for instance, the presence of a salt (NaCl) as an

aggregating agent. The adoption of larger nanoparticles, or of a regularly-shaped

metal matrix may lead to much more predictable behavior of the enhancement

factor, but the choice of the period of the structure must be tailored for a specific

application. The same amino acids in solutions of different pH are currently under

analysis; furthermore, substrates prepared by lithography are being tested in order

to obtain a more uniform substrate and more uniform spectra. Other spectral

distortions could be resolved by using a polarization-resolved device, which would

help to correlate the intensity of a selected Raman band with the direction of the

exciting electric field. Other procedures to address the problem of reproducibility

related to the SERS effect have been proposed, such as averaging of SERS spectra,

using a low magnification objective lens [IV.38] or increasing the colloidal

concentration [IV.31]

Considering the limits and the possible improvements, the current setup showed

that a meaningful analysis of amino acids and peptides present in trace

concentrations can be performed even in absence of advanced laboratory

instruments. The aim of this work was to demonstrate the possibility of adapting a

portable Raman instrument to the SERS analysis of simple organic molecules: this

task was accomplished with careful optimization of the most accessible parameters,

such as the optical components and the fabrication of the metal substrate. The

choice of suitable optics combines the flexibility given by optical fibers to the high

resolution and light collection provided by a microscope objective; the adoption of

131

easy-to-prepare metal colloid must be seen as a preliminary medium to test the

SERS effect on different materials, and it can be replaced by more application-

focused substrates in the future. A more extensive test of both the dry technique and

the portable instrument is planned: if positive results are confirmed, application of

SERS as an in situ diagnostic tool for archaeological and conservation researchers is

foreseen [IV.1]. An important challenge that remains to be investigated is to what

extent one can extract information concerning the nature of archaeological organic

or bio-organic residues through SERS analysis. Searching for specific spectral

signatures that can be correlated with certain materials and/or exclude the presence

of others is a key issue. As indicated above, the use of special preparations of

nanoparticles could potentially lead to SERS analysis based on material specific

enhancement. Additionally the use of chemometrics methods may turn out to be of

importance in screening among different types of residues [IV.39, IV.40]

132

Frequency (cm-1) Frequency SERS (cm-1) Proposed Assignment Bibliographic source

455 480 In phase (C–C–C) [IV.29]

523 521 τ NH3+ , τ NH2) [IV.29]

614 602 In phase (O–CO) [IV.29]

670 662 ω COO ) [IV.29]

721 721 ρ CH2) [IV.29]

796 808 ρ NH2) [IV.29]

898 918 skeletal ν CC , ν CN) [IV.29] [IV.3]

971 964 ρ CH2) [IV.29]

1000 1007 ω CH2) [IV.29]

1050 1050 ω NH3) [IV.29]

1071-1089 1081 ν CN [IV.3]

1138 1130 ρ NH2) [IV.29]

1219 1213 ω CH2) [IV.29]

1315 1314 CH2 ,τ CC [IV.3]

1360 1349 τ CH2) [IV.29]

1442 1444 CH2) [IV.3]

1602 1583 NH2) [IV.29]

1643 NH2) [IV.29]

1703 ν COO ν C=N , NH3+) [IV.29]

Table IV.1. Frequency shift and relative assignment for the detected Raman and SERS bands of L-

Arginine. In this and in the next tables, - deformation, ω - wagging, ρ - rocking, τ - twisting, ν -

stretching.

133

Frequency (cm-1) Frequency SERS (cm-1) Proposed Assignment Bibliographic source

416 410 CCN [IV.9]

450 451 τ NH3) [IV.9]

543 530 CCO [IV.9]

644 644 ν CS CCO [IV.9] [IV.3]

682 660-686 ν CS CCO [IV.9] [IV.3]

719 738 ν CS [IV.9] [IV.3]

767 777 ν CS [IV.9]

804 805 CCH [IV.9]

877 860 ν CC CNH [IV.9] [IV.3]

951 950 ρ CH3) [IV.9]

986 980-987 ν CN [IV.9]

1032 1028 ν CC [IV.9]

1040 ν CN [IV.9]

1071 1068-1078 ρ NH3) [IV.9] [IV.3]

1121 1128 ρ NH3) [IV.9]

1150 1150 CCH [IV.9]

1180 1186 CCH [IV.9]

1250 1243 CCH SCH [IV.9] [IV.3]

1260 1264 -1280 CCH SCH [IV.9] [IV.3]

1322 1318 SCH CH3) [IV.9] [IV.3]

1353 1362 1365 CH2) [IV.9] [IV.3]

1412 CC ν CO [IV.9]

1418 1425 CH3) [IV.9] [IV.3]

1445 1450 ν CC [IV.9] [IV.3]

1510 1506-1513 ν CNH [IV.9]

1628 1623 NH3) [IV.9]

Table IV.2. Frequency shift and relative assignment for the detected Raman and SERS bands of L-

Methionine

Frequency (cm-1) Frequency SERS (cm-1) Proposed Assignment Bibliographic source

622 644 ν Ring [IV.30]

855 858 ν CC – Ring) [IV.30]

913-953 931 C-COO- ) [IV.30,IV.33]

1003 1003 CC – Ring) [IV.30,IV.33]

1036 1031 ν CH , ν Ring [IV.30,IV.33]

1128-1187 1151 CCN asymm [IV.33]

1215 1205 ν Phenyl group [IV.30]

1340 1357 COO-) [IV.30,IV.33]

1411 1418 COO-) symm [IV.33]

Table IV.3. Frequency shift and relative assignment for the detected Raman bands of L-Phenylalanine

134

Frequency (cm-1) Frequency SERS (cm-1) Proposed Assignment Bibliographic source

447 447 ν OH-) [IV.34]

622 622 CS [IV.28]

660 644 CS [IV.28][IV.34]

678 678 CS [IV.28]

737 COO-) [IV.28]

774 765 COO-) [IV.28][IV.34]

811 795 ν COO-) [IV.28]

830 830 C-CN) [IV.28]

920-932 925 C-COO-) [IV.28]

1039 1050 CN [IV.28]

1285 1281 Amide III [IV.28]

1367 1367 COO-) [IV.34]

1445 1448 CH [IV.34]

1632 1634 Amide I [IV.28]

Table IV.4. Assignment for some of the observed Raman bands of Glutathione

135

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CHAPTER V

CONCLUSION.

The common goal of the reported work is to pioneer a new generation of Raman

spectrometers, particularly suitable for the analysis of works of art, thanks to the

mapping approach, but which could also find application in geology or security

controls, for example. The knowledge of the scattering phenomenon on which

Raman spectroscopy is based, together with an in-depth study of the light path

helped us in the determination of the parameters which best fitted the requirement

of a good collection of the signal; most of all, the know-how of a research group

which is well-trained in the application of optical methods of analysis for the study

of Cultural Heritage has guided the construction of an instrument that could match,

as far as possible, the need for a new diagnostic tool. The combination of a near-

infrared laser, which supplied a small but rather powerful light source without the

risk of exciting excessive luminescence, an optical system tailored for the particular

application, the scanning mirrors and the flexibility that the optical fibers give to the

instrument’s layout result in a ductile mapping spectrometer, which covers a niche

in the applications of Raman spectroscopy that was previously unfulfilled.

The Remote Raman Scanner developed as part of this thesis proved to be a suitable

non-invasive diagnostic instrument on a variety of different samples. Tests of

traditional pigments (lead white, ultramarine blue, gypsum and vermilion) on the

model panel, on luminescent white pigments and a modern dye-based yellow

pigment provided good spectra which could be used for material identification,

even though the adoption of a stand-off layout radically decreases the collection

cone of the instrument with respect to a traditional Raman set-up. The result is that

good quality spectra often require acquisition time of the order of a few minutes,

and consequently the background affecting the baseline of the spectrum can be

significant. Nonetheless, the identification of the most intense bands of almost all the

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studied inorganic materials was unambiguous. Moreover, the method for

reconstructing the spectral maps of the target surface, whose application on the

study of minerals has been reported, and is virtually independent of baseline

distortions, thanks to the interactive subtraction of the baseline.

The reconstruction of the intensity profile of the distinguished Raman bands on the

surface under the field of view is the main analytical tool offered by this instrument.

Even if the raster scan of the selected area might be the default choice for probing a

sample, this time-consuming procedure makes the selective analysis a preferable

option: the flexible mapping of the mineral, reported in section III.7 shows how

meaningful information can be extracted by a selection of points, without the need

for a full scan of the area of interest. The multi-analytical approach exploited for the

analysis of the model panel (paragraph III.3) permitted to further limit the number

of needed acquisitions, thanks to the preliminary analysis carried out with

reflectance imaging and X-Ray fluorescence. Of course, the analysis of a surface

depends on the particular application; for this reason, the software control allows a

multiple operation mode (single shot, multiple points with a fixed exposure time,

multiple points with automatic exposure) in order to fulfill specific working

requirements.

The mapping mode has not been applied to the study of the Grillo telephone,

because the spectra collected from different positions on its surface did not show

any significant change. The instrument did not provide any information other than a

confirm for its chemical composition, though, the possibility of performing this

analysis without moving the sample along the field of view plane, and, especially,

along the focal axis, would have been impossible without the peculiarity of Remote

Raman Scanner. The analysis of three-dimensional objects is, thus, possible in a

particularly easy mode.

An application of the system to the analysis of works of art in situ has yet to be

carried out and it is hoped that this will occur in the near futureNonetheless, the

analysed samples and materials have not been treated before the measurement, nor

particular adjustments have been taken when positioning them in front of the

collecting lenses: the long depth of field of the optical system, together with the

robust assembly of the probe give us a solid confidence that the performance on site

would not be so much different from those obtained in laboratory. On the other

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hand, the drawback of the non-contact approach is the need for working in the dark.

The possibility to test two other mobile Raman instruments, currently off-the-shelf,

revealed that the performances of the Remote Raman scanner, on the shown

samples as well as on other standards, are not inferior to the commercially available

counterparts. Of course, some of the ultimate portable Raman spectrometers

provide a better spectral resolution and they are often packed in a more compact

and lighter housing: however, none of these is able to work in a non-contact mode,

nor can they map a surface without a relative movement of the sample. With respect

to the traditional choice of a microscope objective or a short-focal lens as the

terminal optical feature, the long-focal optical system here described, which focuses

the laser beam on a relatively wide spot and therefore deliveries a lower density of

power on the target, presents a reduced risk of damaging the sample.

The work on the surface-enhanced Raman technique employed for helping the

detection of diluted amino acids aims can be seen as a complementary activity in

applying novel techniques for in situ Raman analysis. Not all the issues of this

analytical method can be solved by engineering work on the measurement device,

therefore it is important to carefully evaluate all the other parameters concerning

the measurement as a whole, before to consider it unfeasible. The application of

SERS is a powerful resource to overcome the detection limit of Raman spectroscopy

with organic materials, especially when, as in the cited case of archaeological

vessels, they are present in very low concentrations. This technique is currently one

of the most popular means for the screening and identification of molecules; many

efforts are directed to the development of new materials as substrates which may

grant reproducible spectra. Together with the improvements in the technology for

the detection of optical signals, the possibilities of developing advanced Raman

instruments and methods are in the air. For this reason, it is essential to be ready to

take advantage of the latest discoveries in order to progress in this interesting and

challenging research field.

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Acknowledgements.

I wish to thank prof. Cubeddu for giving me the opportunity of undertaking this PhD;

I thank the former and the current coordinators of the PhD course, respectively prof.

Ciccacci and. prof. Taroni. All my gratitude goes, of course, to my supervisor, prof.

alentini, and to his team: dr. Comelli and dr. Nevin, first of all, but also dr. ’ ndrea,

dr. Osticioli and all the other members of the research group: prof. Pifferi, prof.

Torricelli, dr. Farina, dr. Bassi, dr. Contini, dr. Spinelli, dr. Dalla Mora. I would like to

remember each occasion in which I have been helped by you but I think there is no

space for 140 pages more.

A deserved acknowledgment to my Greek hosts, prof. Fotakis, prof. Anglos and Dr.

Philippidis, for welcoming me in Crete despite the gloomy period.

I feel very grateful towards the researchers and PhD students who collaborated with

this project, and who permitted me to perform the measurements which are here

reported: thanks to Iacopo Osticioli, Austin Nevin, Ilaria Bargigia, Anna Cesaratto,

Francesca Toja, Emanuela Nolfo, Sabrina Moneta, Lucia Ferrario, Iwona Zmuda-

Trzebiatowska and Kristalia Melessanaki.

This is not only the end of the thesis, but also the end of a wonderful period at

Politecnico di Milano. This acknowledgement, therefore, is addressed to everyone

who made me feel at home there. Thank you, I will remember you all.