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page 1of Confoc al Rama n Microscop y

Origins a battle with scattered lightThe first c onfoc al sca nning mic rosco pe wa s invented in 1955 and pa tente d in 1957 by the Harvard g radua te Ma rvin Mins

was obsessed with resolving the mystery of the human nerve system anatomy which aids in performing an outstand

co mp lex set o f co gnitive functions. Thoug h at tha t time the co mm on shap es of nerve c ells we re g enerally known

co nnec tion sc heme s be twe en them were not m ap pe d. The c ritica l obstac le was that the tissue of the ce ntral nervous s

was solidly packed with interwoven parts of cells. Individual cells were practically indistinguishable from each oth

conventional wide-field optical microscopy because of the excessive scattered light blurring the image. Large amou

sca ttered light w as generated in the wide-field microsco py b eca use the whole sam ple had to b e illuminated at once

(Figure 1). Solving the sca ttered light p roblem b ec am e the d riving force for crea tion of the first c onfoc al microsco pe [1].

Optical design principlesProf. Minsky hypo thesized that all the sca ttered light c ould b e a voided by p reventing it from ente ring the m icrosco pe ob je

in the first instance. According to his vision an ideal microscope would examine each point of the specimen at a time

mea sure the a mo unt of light sca ttered or ab sorbed just b y that po int [1]. This ca n be ea sily ac hievab le by setting a pi

aperture in front of the light source (Figure 2). In this case the small pinhole aperture refocused by the microscope obje

illuminates only a single p oint on a sam ple. Since scatt ered light from all other pa rts of the sam ple d oes not exist a ny mo r

total amount of scattered light is reduced by a few orders of magnitude without affecting the focal brightness.

Confocal Raman MicroscopyGeneral Overview

Figure 1. Sca ttered light problem in the wide-field o ptical reflection m icroscop y. Instead of the idea l reflection (b )

most of the real samp les sca tter light (c). Multiple scattering rays are fairly c ollected by the m icroscop e ob jective

and a dd to the blur in the resulting 2D imag e of the samp le.

Sample

Sampleillumination

Sample

Idealreflection

Sample

Real reflection(with scattered light)

(a) (b) (c )

Figure 2. Difference between conventional

wide-field microscopy (a) where large volume

of sample is illuminated at once and the

confocal microscopy (b) where only a single

spot on a sample is illuminated.

Lightsource

Objective

Sample

Dichromaticmirror

Wide-field microscopy -Large volume illuminated

Sample

(a )

Lightsource

Objective

Sample

Dichromaticmirror

Wide-field microscopy -Large volume illuminated

Sample

(b )

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The sec ond pinhole a perture p laced betw een the microscop e o bjective and the imag e p lane rejec ts the residual sca t

rays originated from a ny ou t-of-foc us po ints on the samp le and func tions as a spa tial filter (Figure 3). Figure 4 show s the sim

schem atic d iag ram of reflec tion c onfoca l microsco pe with the two pinhole a pertures.

Suc h an o pt ica l design offe rs a fe w d istinc tive bene fits. The first pinho le ap erture grea tly red uc es sc at te red (stray) ligh

immensely improves the image quality while the second aperture eliminates any image degrading out-of-focus inform

allows for controllable depth of field and gives the ability to collect series of optical sections from bulk specimens. Wit

optical design, Prof. Minsky was able to realize his dream. Figure 5 demonstrates the stunning difference in image q

betw een the wide-field and co nfoca l types of microsco py [2].

Figure 3. Spa tial filtering with the sec ond pinhole

aperture of the horizontal (a) and vertical (b)

out-of-focus rays.

Objective

Sample

Pinholeaperture

f1

f2

In-focus Out-of-focus(horizontal)

Reflectedlight

(a )

Objective

Sample

Pinholeaperture

In-focus Out-of-focus(vertical)

Reflectedlight

(b )

Figure 4. Simplified scheme of the reflective

confoc al mic roscop e with two pinhole ap ertures.

Lightsource

Objective

Sample

Dichromaticmirror

Pinholeaperture

Pinholeaperture

Figure 5. Difference in quality of micro-images

obtained with conventional wide-field (a - c) and

confoc al (d f) microscopy: fluorescently stained

human me dulla (a, d); whole rabbit muscle fibers

stained with fluorescein (b, e); autofluorescence

in a sunflower pollen grain (c, f). Images are

courtesy of Olymp us America Inc [2].

Confoc al Ram an Mic roscopy General Overview

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Mapping the sampleAll the significa nt improvem ents of imag e q uality in confoc al microsc op y co me w ith a price the nec essity to ma p a sam

obta in the full set o f da ta. The ma pp ing c an b e a rrange d in two ways - by sca nning the light b eam along the sam ple w

system of oscilla ting mirrors or by moving the sam p le itself using an X-Y or X-Y-Z sc anning stag es. There a re a few d istin

techniques of samp le m ap ping.

X-Y op tica l sec tioning (Surfac e sc an )This simple t ec hnique cha racte rizes flat sam ples by ma pp ing a thin 2D layer on the sam ple surfac e which is ca lled a

op tica l X-Y sec tion (Figure 6a ). The t ec hnique is useful for biologica l and pha rma ce utica l sam ples laid unde r a c over g las

ge ology a nd ma terial scienc e fo r structural and che mica l ana lysis of p olished thin-sec tions and cut -off sec tions.

X-Z c ross-sec tioning (Dep th profiling)

Confocal microscopy can be used as a powerful non-destructive tool to obtain information about a samples 2D cross-se

in X-Z direction (Figure 6b). Applications of this technique include in vivo biopsy tests and non-destructive analysis of ma

internal structure and integrity.

Z-series (X-Y-Z sc an)

When a 3D structure of a bulk sam ple should b e revea led o ne c an eng ag e in a ted ious task of ta king m ultiple X-Y optica l

sec tions along t he Z-axis (Figure 7a). The resulting informa tion is transferred into softwa re to rec onstruc t the de ta iled 3D im

The t ec hnique is the ba ckb one of c onfoc al m icrosco py and is extrem ely useful for ana lysis of p olymers, nano -mat

integrated circuits, p harma ce utica l sam ples and biologica l ob jects. The first 3D ima ge of a huma n ne ural tissue wa s succ e

ob ta ined b y Prof. Minsky at the end of the 1950s.

Surfac e profiling (Auto -foc using)

In princ iple it is po ssible to reco nstruct a 3D surfac e o f a sample f rom the X-Y-Z sc an . Though, if one is interested in ob ta ining

a surface profile it can be done much faster using an auto-focusing technique. While scanning the sample surface the

po int microsco pe ob jective is mo ving up a nd d ow n to foc us exac tly at the sam ple surfac e (Figure 7b). The tec hnique rese

a profilometer functioning but confocal surface profiling can reveal not only morphological but also detailed che

informa tion ab out the sam ple.


(a )

Single X-Y optical section (2D)

X

Y

(b )

Single X-Z optical section (2D)

X

Z

(a )

Figure 6. 2D mapping: single X-Y (a) and X-Z (b) optical sections on a sample.

(b )

X

Y

Z

Single X-Y-Z optical profile (3D)

Objective

Figure 7. 3D mapping: multiple X-Y-Z optical sections (a) and a single X-Y-Z profile (b) obtained in auto-focus mode.

Multiple X-Y-Z optical sections (3D)

X

Y

Z


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The po we r of confoc al spec tral mea surementsOne o f the ultimate go als of a ny ana lytica l technique is to a chieve t he b est p ossible differentiat ion. In terms of m icrosco py

this means the ability to distinguish between sample details and features of interest as clearly as possible, by somehow

differentiating them from the ambient background. Microscopy has a rich history of finding and developing numerous

dyes, fluorophores and labels to stain target sample features in order to differentiate them from the rest of the sample

Howe ver, the a rsena l of staining a ge nts is limited and som e p roperties and structures are d ifficult to study with c onvent iona

analytical techniques due to their inability to chemically differentiate materials with sufficient spatial resolution, withoutda ma ge or preferential solvent w ashing. This is whe re the true p owe r of c onfoc al m icro-spe ct rosco py unleashes itself.

It is po ssible to co llec t a signal from a single po int on a sam ple, d ispe rse it into a spe ct rum using a spe ct romete r and de tec t

the spectrum using a multi-channel detector such as CCD or PDA (Figure 8). In this case, instead of obtaining trivial

cumulative information about the spot signal intensity we can obtain a signal spectrum which can be transformed into

detailed information about the chemical composition of the given spot on a sample. In other words, we just added a

principa lly new dime nsion in our microsco py m ea surem ents giving us an op po rtunity to differentiate the struct ures neve

dete cta ble before, based o n new information c ontained in the spec tra.

Data Cube sThe c ollec tion of spe c tra taken from m ultiple spo ts on the sam e sam ple is ca lled a Data Cub e be ca use for a simple 2DX-Y scan the data is represented by a 3D array (cube) where X and Y dimensions hold spatial coordinates and the Z-

dimension stores the spe ct ral information (Figure 9). A 3D spe ct ral sca n is represente d by a 4D Data Cub e w hich include s

three spa tial dime nsions plus one spe ct ral dimension. The a dd itiona l spe ct ral dimension in Data Cub es provides a po werfu

too l for differentiation o f internal sam ple structure.

Huma n mind is unab le to c om prehend de nse 3D and 4D arrays of d ata . Therefore, the reco nstructing softw are ha ndling

the Dat a C ube s should ha ve a po tent d ata mining functiona lity. The useful informat ion from the m ulti-dimensional da ta

structures is extrac ted by slicing them along any of spatial o r spe ct ral co ordinates.

Figure 8. Optical scheme for confocal spectroscopy

including confocal microscope, spectrometer and

multi-channel CCD detector.Laser

MicroscopeObjective

Sample

Dichromaticmirror

Pinholeaperture

Pinholeaperture

Spectrometer

CCDDetector

Focusinglens

Figure 9. Structure of a 3D Data Cube : X and Y dimensions

store spatial information, while Z-dimension stores spectra

information from eac h spatial spot.

Y

Z

XWavelength,nm


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Confocal Ram anThe spe ct ral informa tion in co nfoc al microsco py c an b e ob ta ined through d ifferent tec hniques such a s Ab sorption

Reflec tion, Transmission, Emission, Phot oluminesc enc e, Fluoresc enc e o r Ram an spec trosc op y. Amo ng t hose tec hnique s

confocal Raman holds a special place. Raman spectroscopy as such is based on the inelastic scattering of

mono chrom atic light when the freq uenc y of phot ons cha nge s upo n interac tion with a sam ple. The pho tons of the lase

light are absorbed by the sample and subsequently reemitted. Frequency of the reemitted photons is shifted up or down

in com pa rison w ith the original monoc hrom atic freq uenc y, which is known a s the Ram an e ffec t. The Ram an shift p rovide sinformation about vibrational and rotational energies of molecular bonds.

It wa s realized that Ram an spe ct rosco py w as a c onvenient p robe o f the vibrational ene rgy levels within a mo lecule which

ea sily provides mo lecular finge rprints. Anothe r unique ad vanta ge of Ram an spec trosco py is it ca n b e used to selec tively

excite a needed portion of the molecule by changing the excitation wavelength. On top of that Raman spectroscopy

do esnt req uired any sam ple p repa ration, sam ples are not de stroyed, wa ter ba nds are usually sma ll and ea sily subtrac ted

and Ram an spec tra usually conta in sharp b and s tha t a re c harac teristic of t he spe cific m olecular bo nds in the samp le. The

intensity of the ba nds in a Ram an spe ct rum is prop ortiona l to the c onc entration of the co rrespo nding m olec ules and thus

ca n be used for qua ntitative a nalysis.

In biological samples, such as cells and tissues, infrared spectra often show broad spectral features which can give

informa tion reg arding c ellular co mp onent s. How ever, Ram an spe ct ra give this informa tion as well as more d eta iled

informa tion regarding the co nstituents of the se spe c ific co mp onent s allowing go od spe cificity for qualitative ana lysis and

for disc rimina tion a mo ng similar ma terials.

The latest trend in high-end co nfoc al Ram an microsco py instrumenta tion is mo dular system s de signed for ea sy

custom ization of t heir com po nents and therefore p roviding the be st p ossible results in terms of c hemic al d ifferentiation and

sensitivity. One of the best examples is Princeton Instruments modular system. It includes confocal microscope which can

be co upled directly or fiber-op tica lly to the e ntrance slit of a Triple spe c tromete r eq uippe d w ith a multi-cha nnel CCD

ICCD o r InGa As PDA d etec tor (Figure 10). The system is de signed to hand le a wide range of e xcitation w ave lengths

includ ing tuna ble laser sourc es. The 3D Da ta Cub e o bt ained with this system is show n in Figure 11.

Figure 10. Modular c onfoc al Ram an system w ith

Triple spec tromete r from Prince ton Instruments

(dete cto r is not shown): 1 mic rosco pe; 2 X-Y

stage; 3- microscope adaptation optics; 4

excitation beam optics; 5 emission beam

optics; 6 mac ro-samp le c hambe r; 7 switch

between micro- and macro- options, includes

optional optica l elements; 8 - triple

spec tromete r Ac ton TriVista777; 9 - entranc e

slits; 10 switch between triple and single

modes.

Figure 11. 3D Data Cube of a solid micro-

particle obtained with Princeton Instruments

confoc al Rama n system.


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Confocal Ram an App licationsClear ad vanta ge s of c onfoc al Ram an microsco py a ssured its wide-spread proliferation into m ultiple a reas of industry and

academic research. Partially, it has happened due to explosive growth of Nanoscience. Characterization of

heterogeneous nano-systems continues to grow in importance and impacts key applications in the field of

nanotechnology (molecular electronics, nanosensors, nanotubes and nanowires), material science (phase segregated

systems) and catalysis (single site catalysts). One of the hot subjects in this area is characterization of carbon nanotubes

(CNT) the unique na nostructures with rema rkable m ec hanica l and elec trica l prope rties and significa nt p ote ntial for futureinnovations. Confoc al Ram an m icrosco py is ab le to c ap ture the spa ce distribution o f CNT with different elec tronic

structures helping to reveal mechanisms controlling the batch synthesis of preferentially metallic or semiconductor

nanotubes. This type o f ana lysis c an not be done with trad itionally used SEM o r TEM m icrosc op y.

Bio-scienc e resea rch and pha rma ce utica l industry is ano ther rap idly growing a pp lica tion area for confo ca l Ram an. As the

instrumentation evolves towards a dedicated biologists tool, the variety and scope of biological applications will only

widen. The unique a dva ntag e that c onfoc al Ram an microsco py need s no sam ple prepa ration and its ab ility to give

improved axial and spa tial resolution over co nventiona l microsco py m ake it p ossible to p erform e xtreme ly deta iled ana lysis

of c ells in their natural stat e. Ma crosco pic resolution allows investigating the c hem istry of individua l ce lls and ma pp ing of

ge nerated imag es. These imag es ca n co ntain full spe ct ral informat ion at ea ch p ixel so tha t the d istribution of c om po nents

within the c ell c an b e visua lized ba sed upo n their Ram an signa ture. This is extreme ly va luab le to resea rche rs as

biochem ical cha nges ca n be observed during a ce lls life c ycle or when a ce ll bec omes dama ged or ca ncerous. Using

confoc al Ram an, the c hanges in a variety of c ells, including ba cte ria and eukaryotes ca n be monitored over time [3] and

co mp arison b etw een he althy and disea sed tissue stat es ca n be ea sily ana lyzed .

Pharma ce utica l industry has spe cific c hallenges for confoc al Ram an in cha racte rizing the structure and distribution of the

active components within surface coatings of medical devices, high-resolution chemical mapping of commercially

distribute d pills and tab lets and hom og eneity tests of c reams and o intments.

ConclusionConfocal Raman microscopy has proven to be an extremely popular analytical technique with the set of unique

advantages such as:

- No preparation of the sample required.

- Great spatial resolution.

- Clear image quality.

- Outstanding chemical differentiation.

- Ability to p erform 3D ma pp ing of b ulk sam ples.

The te c hnique is useful in multiple a pplica tion a reas of p hysica l and life sc ienc es as well as industry and has grea t

pe rspe ct ives for long-term g rowth.

The latest trend in high-end co nfoc al Ram an microsco py instrumenta tion is mo dular system s de signed for ea sy

c ustomizat ion suc h as the o ne from Princ eto n Instruments. Suc h system s provid e the be st possible c hem ica l differentia tion

and sensitivity.

References1. M. Minsky. Sc anning , V.10, pp 128-138, 1988

2. http:// www .olympusco nfoca l.co m/ theory/ co nfoca lintro.html

3. Xie C, Che n D, Li YQ: Rama n sorting a nd ident ific at ion of single living m icro-orga nisms with op tica l tw ee zers. Op t. Lett .

2005, 30:1800-1802

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