basic spectroscopy

12/4/2014 Basic Spectroscopy

http://www.photobiology.info/Nonell_Viappiani.html 1/24

BASIC SPECTROSCOPY

Santi Nonell1 and Cristiano Viappiani2

1 Institut Quimic de SarriaUniversitat Ramon Llull

Via Augusta 390, 08017Barcelona, [email protected]

2 Dipartimento di Fisica Universita degli Studi di Parma

Viale G.P. Usberti 7A, 43100 Parma, [email protected]

1. What Is Spectroscopy?

The term "spectroscopy" defines a large number of techniques that useradiation to obtain information on the structure and properties of matter.The basic principle shared by all spectroscopic techniques is to shine abeam of electromagnetic radiation onto a sample, and observe how itresponds to such a stimulus. The response is usually recorded as afunction of radiation wavelength. A plot of the response as a function ofwavelength is referred to as a spectrum.

This chapter gives an overview of the spectroscopic techniques mostcommonly used in photobiology research. We have restricted ourselvesto include only those techniques that use ultraviolet or visible light as theprimary stimulus. Also, with the newcomer student in mind, we havechosen to concentrate on describing the principles and main applicationsof the techniques, keeping the discussions of technical details and thenumber of equations to a minimum.

2. What Do Photobiologists Use Spectroscopy For?

Photobiologists use a number of spectroscopic techniques to understandhow photobiological processes occur. This involves in the first placeidentifying the primary photoactive molecular entity whosephotoexcitation by the absorption of light energy triggers the biologicaleffect. A fundamental property of such entities is their absorptionspectrum, which describes their ability to absorb light of differentwavelengths. Determining the absorption spectrum of a photoactiveagent is the first step in understanding the photobiological process inwhich it participates.



Equally important is the identification and characterization of all othermolecular entities involved in the process: excited states, reactiveintermediates, and photoproducts. Spectroscopic techniques are also ofgreat help for this, and even elusive species whose lifetime merely spansa few tens of femtoseconds (1 femtosecond is 1015 seconds or onebillionth of one millionth of a second) can be studied to a high level ofdetail with spectroscopic tools.

Finally, a sound understanding of photobiological processes requires acomplete knowledge of the molecular mechanism through which theyoperate. The term mechanism refers to the sequence of events thatundergo all participating species, the rates at which they occur, and thefactors influencing such rates. Timeresolved spectroscopic techniquesallow photobiologists to unravel the mechanims of photobiologicalprocesses.

This chapter will describe the most common spectroscopic techniquesavailable to the photobiologist, and will illustrate the kind of informationthat can be gained with these techniques.

3. A General (And Overly Simplified) Photobiology Scheme

Every photobiological process starts with the absorption of light energyby the primary lightabsorbing species M that, as a result, is promoted toan electronic excited state M* of higher energy. Molecules in their excitedstates are metastable species, and eventually return to the ground stategiving back the absorbed energy either as light or as heat. In addition,the energy content in ultraviolet and visible radiation is high enough toalso promote competing molecular transformations of M* that lead to theformation of reactive intermediates I, and eventually the photoproduct Presponsible for the end photobiological effect. The competition betweenexcitedstate decay and chemical transformation is an essential featureof all photochemical and photobiological processes, and their relativerates determine the final outcome of the process initiatiated by theabsorption of light energy, hence the importance in measuring suchrates.

Photobiologists use energy diagrams to visually organize these species,placing them at a height related to their energy (Figure 1). This provesvery useful for visualizing the sequence of steps forming the mechanismand the associated energy flows. Spectrocopic techniques helpphotobiologists determine the energy contents of each participatingspecies and the rate constants of the processes relating them.



Figure 1. The basic scheme of photobiological events: theprimary photoactive molecule M absorbs light and is promotedto its excited state, M*. It then undergoes a chemicaltransformation to one or more intermediate species I, andfinally yields a product P. The vertical position of the bars in thediagram represents the energy content of each species.

4. What Spectroscopies Are Available To The Photobiologist?

Documenting the general photobiological scheme discussed in theprevious section requires the identification and characterization of allparticipating species as well as the determination of the rate constantsfor the different reactions. Different spectroscopic techniques areavailable to fulfill this goal, the following being those most commonlyencountered in photobiology laboratories.

4.1 Groundstate AbsorptionThis technique, explained in more detail in Section 5, is applied to stablespecies, "stable" meaning that their lifetime is at least of a fewmilliseconds (1 millisecond is one thousandth of a second). This referstipically to the primary light absorbing agent and the final products. Thesample is irradiated with a continuouswave (CW) beam of light, and thefraction of light absorbed is determined through transmission orreflectance measurements. Because light absorption sends a moleculetemporarily to an upper excited state, the information provided by thistechnique is the absorption spectrum of the photoactive species (Figure2), the energy difference between their ground and excitedstates, andthe probability for light absorption. Absorption transitions are markedwith straight arrows on the energy diagram.



Figure 2. Absorption Transitions In Groundstate AbsorptionSpectroscopy.

4.2 Transient AbsorptionThis technique is very similar to the previous one except that the speciesprobed are metastable (i.e., transient) excited states and reactionintermediates whose lifetime may range from femtoseconds, for theprimary excited states, to kiloseconds for slow reaction intermediates.The species are promoted temporarily to upper excited states (see Figure3). Measuring the absorption spectrum of a transient species requires theuse of pulsed lasers for generating a measurable concentration of excitedstates, and a second beam of light, CW or pulsed, for probing theirabsorption. In addition to absorption spectra, transient absorptiontechniques also yield the the rate constants of the processes where thesespecies participate.



Figure 3. Absorption transitions in transient absorptionspectroscopy.

4.3 Emission TechniquesEmission techniques measure the electromagnetic radiation emitted upondeactivation of excited states. When such excited states are created bythe absorption of light, the techniques are referred to as fluorescenceand phosphorescence, depending on the nature of the excited state.Some chemical and enzymatic reactions also produce excited states,whose emission is then referred to as chemiluminescence. Emissiontechniques provide emission spectra, excitedstate lifetimes, and rateconstants for the processes where these species participate. Emissiontransitions are also signalled by straight arrows on energy diagrams (seeFigure 4).

Figure 4. Emission transitions in luminescence spectroscopy.

4.4 Photothermal TechniquesIn addition to, and sometimes instead of, emitting light, excited statesand reactive intermediates give off their excess energy through nonradiative deexcitation pathways, causing local changes in temperature,refractive index and volume. Photothermal techniques monitor suchchanges and thus provide information on the processes that originatethem. The typical use of photothermal techniques is for determining theenergy and volume changes accompanying the reactions of the excitedstates and reactive intermediates (see Figure 5). Thermal or nonradiative transitions are conventionally marked with wavy arrows on theenergy diagrams.



Figure 5. Radiationless transitions in photothermalspectroscopies.

4.5 Timeresolved vs. steadystate techniques.Optical (absorption and emission) and thermal spectroscopies rely on thecreation of excited states and reactive intermediates through theabsorption of light, which leads to two fundamentally differentapproaches: in steadystate spectroscopies the samples are continuoslyirradiated with a beam of light; excited states are continuously createdand eliminated and eventually a steadystate is reached where theirconcentration remains constant. This facilitates the measurement ofweak signal levels at the expense of loosing kinetic information. Steadystate spectroscopies are best applied to the measurement of absorptionand emission spectra. On the other hand, timeresolved spectroscopiesrely on the irradiation of the sample with a light source whose intensityfluctuates as a function of time. The simplest example of this is a pulsedlaser, which emits flashes of light. Each flash creates a burst of excitedstates, whose evolution can be monitored as a function of time. Timeresolved spectroscopies provide kinetic information at the expense of alower sensitivity compared to steadystate techniques.

5. GroundState Absorption

The absorption spectrum of stable molecules, such as the primaryphotoactive species or the final products, is most conveniently recordedusing a steadystate spectrophotometer. Modern instruments consist of acombination of light sources covering the UV and visible ranges, amonochromator to isolate a narrow wavelength range, a samplecompartment, and a detection system (Figure 6). Transparent samplessuch as films or solutions are best measured in transmission mode, theinstrument measuring the intensity of light transmitted by the sample.Opaque or highly scattering samples such as solids or suspensions arebest measured in reflectance mode.



Figure 6. Typical optical layouts for absorptionspectrophotometers. Left: In diodearray spectrophotometers,the spectrum is obtained at once. Right: in single detectorspectrophotometers, the amount of light transmitted (orreflected) is detected one wavelength at a time.

The transmittance of a transparent sample is defined as the fraction oflight transmitted by the sample when it is irradiated with a beam of light.It is determined by measuring the intensity (more properly called radiantpower) of the incident and transmitted light beams. A plot of thetransmittance vs the beam wavelength is called a transmission spectrum.Photobiologists usually prefer to work with absorption spectra (Figure 7),where the related quantity absorbance is plotted instead. The absorbanceof a sample is defined as the negative logarithm of its transmittance:Absorbance = log (Transmittance). For a pure substance, theabsorbance of a sample at a given wavelength is directly proportional toits concentration.



Figure 7. Absorption spectrum of (a) chlorophyll and (b) Fe(II)protoprophyrin IX, the prosthetic group of heme. The sharpabsorption of chlorophyll in the red part of the spectrum makesgrass green, and its lack in heme makes blood red.

The reflectance of an opaque sample is likewise defined as the fraction oflight reflected by the sample when it is irradiated with a beam of light. Itis determined by measuring the intensity (radiant power) of the incidentand reflected light beams. A plot of the reflectance vs the beamwavelength is called a reflectance spectrum.

Photobiologists use absorption (or reflectance) spectra to: * Identify thewavelengths that a sample can absorb and the relative probabilities ofthe absorption transitions at each wavelength.

* Identify the molecular species responsible for the photobiological effectunder study.

* Determine the energy of the molecule's excited states.

* Study the interactions between species, e.g., a drug with a hostprotein.

6. Transient Absorption

6.1 The pumpandprobe approachTransient absorption techniques rely on the use of a probe beam thatinterrogates the sample before and after excitation with a pulsed laser(pump beam), and monitors absorbance changes either at a selectedwavelength (kinetics) or simultaneously at several wavelengths(transient spectra). These methods are collectively referred to as pumpandprobe methods although, depending on the time scale, the transient



absorbance techniques may have very different experimental layouts.These techniques allow, in general, to study the disappearance of theexcited state M*, the formation of the reactive intermediates I, and theformation of photoproducts P.

The pump laser must excite the sample at a wavelength where groundstate absorption occurs (Section 5). The laser pulse width must be muchshorter than the time constant of the reaction under investigation.

The probe beam is generally a broadband light source in the UVvisNIRspectral range, where transient electronic transitions occur. The probelight source is normally a CW or pulsed lamp for nanoseconds (1nanosecond is one billionth of a second) or longer time scales, while apulsed laser is used for higher time resolution.

The experimental parameter of interest is a change in sample absorbance( A), obtained from the change in transmitted light intensity of the probebeam before, I(t<to), and after, I(t), laser excitation, according to thefollowing equation:

A(t)= log [I(t)/I(t<to)].

Photobiologists use transient absorption to:

* Identify reaction intermediates from their spectral features

* Assess the reaction mechanism by detecting spectrally distinguishablereaction intermediates

* Determine the rate constants for each kinetic step

* Determine activation parameters from temperature dependence of rateconstants

* Determine reaction quantum yields in favourable cases, whereextinction coefficients of reaction intermediates are known.

6.2 From milliseconds to hours: steadystate spectrophotometersWhen very slow reactions (time constants in the range of minutes orlonger) are to be monitored, the changes in the absorbance of thesample following photoexcitation by the pulsed laser can be followedusing conventional steady state spectrophotometers (Section 5),provided that the absorption spectra can be taken on time scales muchfaster than the characteristic time of the reaction under investigation.Spectrophotometers also allow one to follow the absorbance at a selectedwavelength as a function of time, thus providing a kinetic time course forthe photoinduced reaction.

For faster reactions (down to a few milliseconds) multichannel detectors,such as charge coupled devices (CCDS), and photodiode arrays (PDA)are normally used to follow the time evolution of the absorptionspectrum. In these applications, a CW broadband light source is used to



probe the absorption of the sample. The polychromatic beam is passedthrough a spectrograph to measure absorption spectra as a function oftime. (Figure 6, left) Specialized setups also allow to follow theabsorbance at a selected wavelength as a function of time.

6.3 From milliseconds to nanoseconds: nanosecond laser flashphotolysisAbsorbance changes, from milliseconds to nanoseconds, followingexcitation with a nanosecond pulsed laser can be monitored using a CWlight source such as a Xe arc lamp (Figure 8). A fast shutter exposes thesample to the probe beam shortly before the laser pulse hits the sample,and is closed after the end of data collection to prevent itsphotobleaching. (Figure 9) The polychromatic beam is passed through aspectrograph, either to select the wavelength at which the reactionkinetics are monitored or to measure transient spectra as a function oftime (Figure 10). Examples of timeresolved spectra and rebindingkinetics are displayed in Figures 11 and 12. The spectral and kineticinformation allow to identify reaction intermediates and to retrieve rateconstants.

Figure 8. Essential optical layout of a nanosecond laser flashphotolysis setup. A CW lamp beam monitors changes ofabsorbance (or reflectance) in a sample after flashphotoexcitation with a pulsed laser.



Figure 9. Time profile of probe light intensity transmitted bythe sample. At time zero, the shutter is closed and the detectorsees no light. When the shutter is opened the light transmittedby the sample is seen by the detector. The changes in intensityinduced by the pulsed laser are highlighted as a blue signal. Atthe end of the experiment the shutter is closed again toprevent extensive photodegradation of the sample.

Figure 10. Typical optical layout of the detectors used innansoecond laser flash photolysis. The spectrograph can select



a specific wavelength and allow monitoring the absorbancechanges at that wavelength. Alternatively, a spectral range canbe monitored by a multichannel detector such as a (CCD) andallow the determination of transient spectra at selected timedelays.

Figure 11. Timeresolved changes in absorption spectrumfollowing photodissociation of carboxyhemoglobin. Ananosecond laser pulse photolyzes the COFe bond leading tochanges in the heme absorption spectrum in the Soret region.The ligand is then rebound by the heme, with the complexkinetics shown in the plot, extending over several orders ofmagnitude in time. The protein is embedded in a silica gel toprevent quaternary switching from the R to the T quaternarystructure after photodissociation.



Figure 12. Time course of the change in absorbance at 436nm, following nanosecond photodissociation of carboxyhemoglobin. The change in absorbance reflects the kinetics ofcarbon monoxide rebinding to the heme Fe.(see Viappiani, C.et al. Proc. Natl. Acad. Sci. USA 2004, 101, 1441414419. fordetails).

6.4 The subnanosecond domain: the twopulse pumpandprobetechniqueWhen absorbance changes are to be monitored in the subnanoseconddomain, the probe beam is obtained by a second broadband laser pulse,hitting the photoexcited sample at a proper time delay (Figure 13).

Figure 13. Scheme for subnanosecond pumpandprobemethods.

Modern pump lasers emit pulses of approx. 100 femtoseconds, which are



tuneable across broad wavelength ranges, and have a high repetitionrate (80 MHz). The probe beam is normally obtained by splitting aportion of the pump beam, which is then focussed onto suitable materialsgenerating spectrally broad pulses in the region of interest. Kineticinformation is obtained by adjusting the time delay between pump andprobe pulses. By probing the photoexcited sample as a function of timedelay of the probe pulses, timeresolved spectra can be obtained, fromwhich the kinetics at selected wavelengths can be reconstructed. Resultsare similar to those represented in Figures 11 and 12, except for the timescale.

7. Emission Spectroscopies

In emission spectroscopy, a sample is irradiated with a beam of light (theexcitation beam), and the luminescence emerging from the cuvette isrecorded with a suitable detector. Such luminescence is caused by theradiative decay of the excited states created by the excitation beam,which thereby return to their ground state. The emission detector isplaced tipically at right angle to the excitation beam to avoid anytransmitted light. (Figure 14).

Figure 14. Optical layout of a steadystate spectrofluorometer.A light beam produced by a CW lamp is passed through amonochromator to select a particular excitation wavelengththat is then focused onto a sample. The ensuing emission isobserved at right angles, analyzed spectrally with the emissionmonochromator, and recorded by an appropriate detector.

When the excited state has the same spin multiplicity as the ground statethe radiative decay transition is labelled as "spin allowed" and the



emission is referred to as fluorescence. When the spin changes thetransition is said to be forbidden and the emission is calledphosphorescence. From a kinetic point of view, fluorescence emission is afast process, typically in the nanosecond range, while phosphorescencemay last anything from microseconds to hours. In addition,phosphorescence always occurs at longer wavelengths than fluorescence(Figure 15).

Figure 15. Fluorescence (top) and phosphorescence (bottom)spectra of tryptophan in a protein (black) and in solution (red).Notice that phosphorescence occurs at longer wavelengths thanfluorescence. Data are courtesy of P. Cioni, CNR, Pisa (Italy).

7.1 Steadystate emission spectroscopyIn steadystate spectrofluorometers, the sample is irradiated with a CWbeam of light, very much as in absorption spectroscopy. This creates asteady concentration of excited states, and therefore a steady emissionof light from the sample. Excitation and emission monochromators allowthe user to select the wavelengths of the excitation and emission beams.

Emission spectra are obtained by setting the excitation wavelength to aconstant value and scanning the emission monochromator. The recordedemission intensity is then plotted as a function of the emissionwavelength (Figure 16). Alternatively, the emission wavelength is lockedand the excitation monochromator is then swept. This produces anexcitation spectrum. For a pure substance existing in a single molecularform in the sample the excitation spectrum matches its absorptionspectrum, which is very useful to identify the emitting species in amixture. Differences between absorption and excitation spectra indicate



the presence of more than one species in the sample, either due todifferent substances or different forms of the same substance, e.g. anacid and its conjugated base or a monomer and a dimer, etc.

Figure 16. Absorption, fluorescence emission, andfluorescence excitation spectra of a green fluorescence proteinmutant.

The fluorescence (or phosphorescence) quantum yield expresses theprobability of emitting light upon absorption of radiation and is calculatedby dividing the number of light quanta emitted by the number of quantaabsorbed.

Photobiologists use steadystate emission spectroscopy to:

* Assess the ability of a substance to emit light, and the wavelengthswhere this emission occurs. The corresponding properties are theemission quantum yield and the emission spectrum.

* Identify the molecular species responsible for the photobiological effectunder study. This is inferred from the excitation spectrum.

* Determine the energy of the molecule's excited states. The wavelengthof the fluorescence onset gives a good estimation of the energy of thefirst excited singlet state, while that of the phosphorescence onset givesthe energy of the triplet state.

* Study the interactions between species, e.g., a drug with a hostprotein, which often result in changes of the spectrum and/or quantumyield of the emission.

7.2 Timeresolved emission spectroscopyTimeresolved emission spectroscopy provides information on the kineticbehavior of luminescent excited states and intermediates. The optical



layout is essentially identical to that given in Figure 14, but a light sourcewhose intensity changes as a function of time is used instead. Thiscreates a population of luminescent species that also change over timeand therefore renders it possible to monitor the emission decay kinetics(Figure 17). Typical light sources include pulsed or modulated lamps,lightemittingdiodes, or lasers.

Figure 17. Decay of fluorescence (top) and phosphorescence(bottom) of tryptophan in a protein (black) and in solution(red; actually the sample is Nacetyltryptophanamide, NATA).Notice that phosphorescence lasts much longer thanfluorescence. Data are courtesy of P. Cioni, CNR, Pisa (Italy)and T. Gensch, Research Center Jülich (Germany).

Photobiologists use timeresolved emission spectroscopy to:

* Obtain rate constants for processes involving luminescent species.These are in most cases obtained in a straightforward way from thekinetic analysis of the intensityvstime luminescence data.



* Unravel the mechanism of a photobiological process by characterizingthe reactive intermediates and their rates of formation and decay. This isusually achieved from the analysis of timeresolved emission spectra(TRES), where the luminescence is recorded both as a function of timeand wavelength.

* Study the interactions between species, e.g., a drug with a hostprotein. This is particularly useful when such interactions have nomeasurable effect on the emission spectra of host or guest but rather ontheir luminescence lifetimes.

8. Photothermal Techniques

Photothermal tecniques are a group of high sensitivity methods thatmonitor the effects induced in solution after nonradiative relaxation ofexcited states. The basis of the collective term photothermal for thesetechniques lays in the detection of thermal relaxation of excess energyassociated with photoexcitation of the sample. In addition, other nonradiative nonthermal relaxations, such as volumetric changes due tophotoinduced structural changes (isomerization, charge transfer, solutesolvent rearrangement, ...) may give rise to detectable signals. The mostremarkable advantage of these methods is that, through a properanalysis, thermodynamic parameters (energy and volume changes) foreach step of the photoinduced reaction can be obtained. The inherenttime resolution of the methods allows one to also retrieve kineticparameters (rate constants, quantum yields) for the reaction steps.Timeresolved photoacoustics is the technique of choice for reactionssteps with lifetimes between about 10 nanoseconds and 10microseconds. For photoinduced reactions with longer time constants,photorefractive methods are more adequate, since they can accessevents with lifetimes extending to the milliseconds.

Photothermal methods generally apply to optically transparent, lowabsorbing samples, although timeresolved photoacoustics can be appliedto strongly absorbing samples, with dedicated experimental setups.

Photobiologists use photothermal methods to:

* Estimate enthalpy and volume changes for each kinetic step

* Estimate the energy content of reaction intermediates

* Determine the quantum yield of photoinitiated reactions

* Determine the rate constants for each kinetic step

8.1 Timeresolved photoacousticsTimeresolved photoacoustics monitors the pressure changes induced ina sample after nonradiative relaxations following excitation with a(normally) nanosecond pulsed laser. The pressure pulse generally has



two sources, both leading to a volume change of the solution, and relatedwith nonradiative relaxations of the excited state. The first source is thethermal relaxation associated with energy changes accompanyingrelaxation of the excited state and reaction intermediates. The secondsource is due to volume changes associated with structuralrearrangements accompanying each step of the photoinduced reaction.The time evolution of the pressure pulse is monitored in microseconds(one microsecond is one millionth of a second) by a fast piezoelectricmicrophone (Figure 18). Calibration of the setup to retrievethermodynamic and kinetic parameters implies comparison of thephotoacoustic signal with that obtained for a photocalorimetric referencecompound (i.e., a compound that undergoes thermal relaxation with unitefficiency within a few nanoseconds). (Figure 19) The resonantconfiguration of the detector requires the use of numerical deconvolutionanalysis to retrieve kinetic information from experimental signals.

Figure 18. Essential scheme of a time resolved photoacousticssetup.



Figure 19. Photoacoustic signals for degassed acetonitrilesolutions of the photocalorimetric reference compound2,hydroxybenzophenone (R(t)) and benzophenone (S(t)) atroom temperature. Both the amplitude and the shape of thesignal for benzophenone are different from those for2,hydroxybenzophenone. The signal for benzophenone, ofthermal origin, is best described by a double exponential decaywith a fast component (lifetime below ( 10 ns), due to tripletformation, and a slower relaxation (lifetime of ( 6.7microseconds), due to triplet decay.

8.2 Photorefractive techniquesPhotothermal lensing, beam deflection, and grating techniques detectchanges in refractive index upon changes in density (resulting from achange in volume of either thermal of structural origin), in absorbance,and in temperature. When a sample is excited with a light beam, whichusually has a Gaussian shape, being absorbed by the sample, theconcentration of the excited state molecules reflects the pump beamgeometry. The nonradiative relaxations of the excited states heat thesample resulting in a temperature profile, which reflects the Gaussianintensity profile of the exciting laser beam. A decrease in density resultsfrom heating, eventually leading to a decrease in the refractive index ofthe sample. These methods allow one to retrieve enthalpy and volumechanges associated with the relaxation of the photexcited molecules, andthe rate constants of these processes.

Three basic experimental approaches are used to detect the refractiveindex change: the transient lens, the beam deflection, and the transientgrating.

Transient lens. A CW probe beam travels through the illuminated region(by the pump laser) and is expanded by the Gaussian profile of therefractive index. This effect can be detected from the change in lightintensity through a pinhole (Figure 20).



Figure 20. Schematic of the experimental setup in transientlens experiments. The pump blue beam generates a change inrefractive index. The red probe beam is expanded by therefractive index profile. A detector placed after a pin holesenses the defocusing as a change in light intensity.

Beam Deflection. The CW probe beam is not perfectly concentric withthe pump beam. The spatial profile of the refractive index change resultsin a deflection of the probe beam. A position sensitive detector monitorsthe displacement of the probe beam (Figure 21).

Figure 21. Schematic of the experimental setup in beamdeflection experiments. The pump blue beam generates achange in refractive index. The red probe beam is deflected bythe refractive index profile. A position sensitive detector sensesthe deflection.

Transient Grating. Two pump laser beams with parallel polarization arebrought into the sample at an angle and from their interference pattern amodulation of the excitation intensity results. The refractive indexchanges originating from the photoinduced processes reflect thismodulation, and can be monitored by a third laser beam, which isdiffracted to an extent depending on the grating properties. Anadvantage of this method is that it can access subnanosecond events



(Figure 22).

Figure 22. Schematic of the experimental setup in transientgrating experiments.

The rate of formation of the density lens (≈ 107 s1) sets the timeresolution in transient lens and beam deflection. On the other hand, thetechniques are very sensitive in detecting slower kinetics, extending tomilliseconds. In order to retrieve quantitative thermodynamic informationfrom the lens and beam deflection signals, a photocalorimetric referencemust be used to calibrate the instrumental response, in essentially thesame way as already described for the photoacoustic signals. Theadvantage of these methods is that they require no deconvolutionanalysis to retrieve the kinetic information.

9. Concluding Remarks

The photobiologist of the 21st century might rather be called a molecularphotobiologist in that the molecular approach provides the most accuratealbeit complex description of photobiological phenomena. Spectroscopyoffers a wonderful window into such a molecular world, and provides uswith a unique yet powerful set of tools to explore the intricacies ofphotobiological phenomena. Techniques that were rather sophisticatedjust a decade ago are now being routinely used in many laboratoriesworldwide, revealing a wealth of data which is changing the way weunderstand nature.

10. Further Reading

Nanosecond Laser Flash PhotolysisBonneau R., Wirz J., Zuberbuehler A.D. (1997) Methods for the analysisof transient absorbance data, Pure & Appl. Chem. 1997, 69, 979992.



Chen E., Goldberg R. A., Kliger D.S. Nanosecond timeresolvedspectroscopy of biomolecular processes, Ann. Rev. Biophys. Biomol.Struct. 26, 327355.

Tetreau C., Lavalette D. (2005) Dominant features of protein reactiondynamics: Conformational relaxation and ligand migration, Biochim.Biophys. Acta 1724, 411424.

Abbruzzetti S., Bruno S., Faggiano S., Grandi E., Mozzarelli, A.,Viappiani, C. (2006) Timeresolved methods in Biophysics. 2. Monitoringhaem proteins at work with nanosecond laser flash photolysis,Photochem. Photobiol. Sci., 5, 11091120.

Picosecond Transient AbsorbanceCerullo, G., Manzoni C., Luer L., Polli, D. (2007) Timeresolved methodsin biophysics. 4. Broadband pumpprobe spectroscopy system with sub20 fs temporal resolution for the study of energy transfer processes inphotosynthesis, Photochem. Photobiol. Sci., 6, 135144.

Groot, M.L., van Wilderen, L.J.G.W., Di Donato, M. (2007) Timeresolvedmethods in biophysics. 5. Femtosecond timeresolved and dispersedinfrared spectroscopy on proteins, Photochem. Photobiol. Sci., 6, 501507.

Emission SpectroscopyLakowicz, J.R. (2006) Principles of Fluorescence Spectroscopy, 3rd ed.,Kluwer Academic/Plenum Publishers, New York.

Becker, W. (2005), Advanced TimeCorrelated Single Photon CountingTechniques, Springer, Germany.

Time Resolved PhotoacusticsBraslavsky, S.E., Heibel, G.E. (1992) Timeresolved photothermal andphotoacoustics methods applied to photoinduced processes in solution,Chem. Rev. 92, 13811410.

Gensch, T., Viappiani, C. (2003) Timeresolved photothermal methods:accessing timeresolved thermodynamics of photoinduced processes inchemistry and biology, Photochem. Photobiol. Sci. 2, 699721.

Photorefractive TechniquesSchulenberg, P. J.; Braslavsky, S. E. (1997) Timeresolved photothermalstudies with biological supramolecular systems, in Progress inPhotothermal and Photoacoustic Science and Technology, Mandelis, A.,Hess, P., Eds, SPIE Optical Engineering Press, Washington, pp. 5781.

Terazima, M. (2001) Protein dynamics detected by the timeresolvedtransient grating technique, Pure & Appl. Chem. 73, 513517.

10/16/08



[ TOP ]

basic spectroscopy

Documents