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Time-Resolved Raman Spectroscopy
Time-Resolved Raman Spectroscopy H Hamaguchi, National Chiao Tung University, Hsinchu, Taiwan K Iwata, Gakushuin University, Tokyo, Japan ã 2017 Elsevier Ltd. All rights reserved.
Time-resolved Raman spectroscopy (TRRS) refers to Raman spectroscopy that measures time-dependent phenomena in a time-specific manner along their time course. It possesses the ultrafast capability of laser spectroscopy and the high information content of Raman spectroscopy. TRRS has recently evolved into time- and space-resolved Raman spectroscopy (TSRRS) by the alliance with confocal optical microscopy. Highest achievable time-resolution of TRRS is subpicosecond (limited by the time-energy uncertainty principle, see below) and highest achievable space-resolution of TSRRS is a few hundred nanometers (diffraction limited). The application of TRRS/TSRRS extends tremendously in recent years not only in basic science like physics, chemistry, and biology but also in many industrial, pharmaceutical, medical, agricultural, and health sciences and technologies.1 It can measure a variety of time-dependent phenomena starting from cell division in the minute regime to ultrafast photochemical reactions in the pico- and femtosecond regimes, to provide otherwise unobtainable information based on Raman spectra (molecular fingerprints). In the early days of Raman spectroscopy (1928–60s), mercury lamps were used for Raman excitation and photographic plates were used for detection. Time-resolved measurements were not realistic since long exposure time (tens of minutes to hours) was needed for recording one Raman spectrum. After the introduction of continuous wave (CW) laser sources and photomultipliers in 1970s, sensitivity of Raman spectrometers improved markedly but the recording time was not much shortened (typically tens of minutes) because of scanning monochromators. TRRS was still not practical at this stage. Pulsed lasers and multichannel detectors introduced in 1980s changed the situation drastically. Combination of Q-switched pulsed lasers with intensified photodiode arrays (IPDA) made it feasible to perform TRRS in the micro- and nanosecond time regimes.2 Multichannel detectors facilitated the use of pulsed lasers for Raman excitation as well as Raman measurements without scanning spectrometers. Introduction of amplified mode-locked lasers advanced TRRS to the picosecond regime in 1990s and to the subpicosecond in 2000s. CCD (charge coupled device) detectors replaced IPDA after 1990s to increase the sensitivity of TRRS. The use of a narrow bandpass filter (notch filter) with a single polychromator also made the sensitivity of TRRS much higher in 2000s.3 TRRS with nonlinear Raman effects such as CARS (coherent anti-Stokes Raman scattering) and SRS (stimulated Raman scattering) was also developed after 1990s in the pico- and subpicosecond time regimes. TRRS can be classified into two categories according to time regimes that it measures. One is to look at slow changes in the millisecond or longer time regimes. RRS in this time regime is readily performed with the use of a CW laser source and a CCD detector. A commercial Raman spectrometer is capable of doing this job without much difficulty. The other is to look at changes in the ultrafast time regimes of micro-,
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
nano-, pico-, and subpicosecond. TRRS in these time regimes, ultrafast TRRS, needs pulsed laser sources for excitation and (gated) multichannel detectors for detection. Special setups specifically designed for specific purposes are necessary. Ultrafast TRRS commonly adopts the pump-probe resonance Raman scheme, in which a pump-laser pulse first photo-excites the sample to start the change and then a probe-laser pulse with varied delay times probes resonance Raman scattering from the changing sample. In this regard, TRRS is sometimes called timeresolved resonance Raman (TR3) spectroscopy. The principle of pump-probe experiment is schematically shown in Fig. 1 for nanosecond TRRS of benzophenone in the lowest excited triplet state.
TSRRS in the Hectosecond Regime An example of TSRRS application to fundamental biology is given in Fig. 2, in which a single living fission yeast (Schizosaccharomyces pombe) is measured with time-resolution of 100 s and space-resolution of 250 nm.5 It elucidates the change of molecular compositions that occurs at the central part (marked by asterisks in the figure) of the cell. Fission yeast repeats cell division according to the cycle G2 ! M ! G1/S ! G2 with a period of a few hours. The Raman spectrum of the dividing nucleus at 0 min is dominated by known Raman bands of proteins. As the mitosis proceeds M ! G1/S (6, 11, 24, and 31 min), many strong bands ascribed to phospholipids appear. In the S period (41, 62, and 69 min), the spectra are dominated by polysaccharide bands from the septum. At 72 min, the spectrum shows protein/lipid bands of the cell walls of the two daughter cells. This is the first in vivo Raman observation of the dynamic behavior of biomolecules during the cell cycle. The change indicates clearly that the cell is actually “living” during the Raman measurement.
TRRS in the Micro- and Nanosecond Regimes The first ultrafast pump-probe TRRS experiment was reported in 1976 by Pagsberg et al.6 They excited p-terphenyl by pulse radiolysis with an electron pulse (2 MeV, 30 ns duration) from a Febetron accelerator and probed the generated p-terphenyl anion radical with a flash-lamp pumped dye laser pulse at 481.6 nm (10 mJ, 600 ns). Following this work, many pumpprobe TRRS experiments were reported in the micro- and nanosecond regimes. Pulsed Q-switched lasers, Q-switched Nd:YAG lasers in particular, were most suitably used for both pumping (photoexcitation) and probing. A typical set up for nanosecond TRRS is shown in Fig. 3. As an example of TRRS in the micro- and nanosecond regimes, time-resolved Raman spectra of transient deoxyhemoglobin (human) in the time range of 10 ns to 120 ms are shown
http://dx.doi.org/10.1016/B978-0-12-409547-2.12166-3
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Fig. 1 Nanosecond pump-probe time-resolved Raman spectroscopy of the lowest excited triplet (T1) state of benzophenone.4 The fourth harmonic of a Q-switched Nd:YAG laser (266 nm) was used for pumping benzophenone from the ground (S0) to the lowest excited singlet (S1) state and the second harmonic (532 nm) in resonance with the Tn T1 electronic transition with delay time of 15 ns was used probing resonance Raman scattering from T1 benzophenone. The T1 Raman spectrum (c) is obtained as a difference spectrum between the pump þ probe spectrum (a) and the probe only spectrum (b).
Fig. 2 Time- and space-resolved Raman spectra of a dividing fission yeast cell, whose nuclei are labeled by GFP so that the positions and the number of nuclei are directly seen as fluorescence images (shown on the right-hand side). Asterisks indicate the position of the focused exciting laser beam. The 632.8 nm line of a He–Ne laser was used with 1 mW power at the sample point.
in Fig. 4.7 In this experiment, carboxyhemoglobin is photolyzed to induce the dissociation of CO from the heme and to produce transient deoxyhemoglobin. Resonance Raman spectra of this transient deoxyhemoglobin are observed in the time course of its relaxation. In Fig. 4, time evolution of
iron-proximal histidine stretch frequency is clearly observed showing the submicrosecond structure relaxation of transient deoxyhemoglobin. This experiment, together with similar TRRS experiments, has contributed significantly to the elucidation of the hemoglobin cooperativity mechanism, in which the
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Fig. 3 Block diagram of a typical nanosecond TRRS system in the 1980s.2 The second, third, and fourth harmonics of a Q-switched Nd:YAG laser and outputs from a dye laser pumped by the second harmonic were used for pumping and/or probing. A triple polychromator equipped with a subtractive double grating filter was used for a wide wavelength-range measurement. An intensified photodiode array detector (IPDA) was used for nanosecond time-gated detection to eliminate longer lifetime fluorescence/phosphorescence.
heme ligation states and the protein tertiary/quaternary structures are linked through the strain of the iron-histidine linkage of the heme.
TRRS in the Pico- and Subpicosecond Regimes
Fig. 4 Time-resolved Raman spectra of photolyzed COHb in the time range of 10 ns to 120 ms after photolysis.7 The 405 nm output (10 ns pulse duration) of a nitrogen laser pumped dye laser was used for pumping and the 435 nm output (10 ns) of an excimer laser pumped dye laser was used for probing. The pump and probe pulses were synchronized electronically with varied delay time.
Advancement of laser technology in 1990s has made it possible to produce light pulses in the ultraviolet to infrared region with pulse duration of pico- to subpicoseconds. These light pulses have been used for TRRS in the pico- and subpicosecond regimes. There is a minimum value, however, for the product of the energy width and the time width of a light pulse. A light pulse is well described as a wave packet of electromagnetic waves with different frequencies. The electric field of a light pulse as a function of frequency E(n) is converted by Fouriertransformation to the electric field as a function of time, E(t). The product of the frequency width and time width is analytically calculated for a light pulse with several pulse envelopes including the Gaussian, Lorentzian, and sech functions. These values are readily converted to the frequency–time product for the light intensity which is proportional to the square of the electric field of the light pulse. If the frequency–time product of light pulses matches the product value expected from the Fourier transform, then these pulses are called “Fourier-transform limited pulses.” The wavenumber width full width at half maximum (FWHM, cm1) of a Fourier-transform limited pulse is plotted against the duration (FWHM, ps) for Gaussian and sech2 pulses in Fig. 5.8 For a Gaussian pulse, for example, the wavenumber width is equal to or larger than 4.9 cm1 if
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the pulse duration is 3 ps. The pulse duration should be 3.0 ps or longer if wavenumber width of 5 cm1 is required for the measurement. The limitation imposed by the minimum wavenumber–time product is important when designing a TRRS measurement in a pico- or subpicosecond regime. Conventional pump-probe technique is used for picosecond time-resolved spontaneous Raman spectroscopy. Schematic diagram of a picosecond time-resolved Raman spectrometer is shown in Fig. 6.9 The pump and probe pulses with 2 ps duration for this spectrometer are prepared at two OPAs (optical parametric amplifiers) pumped by the output from a Ti:sapphire regenerative/multipass amplifier. The wavelengths of the pump and probe pulses are independently
Fig. 5 Relation between wavenumber width (FWHM, Dn) and pulse duration (FWHM, Dt) of Fourier-transform limited light pulses with pulse shape of Gaussian (a) and sech2 (b).
tunable. A 4f-filter formed by a pair of gratings and a slit is placed for limiting the energy width of the probe pulse. The intermolecular energy transfer process proceeds in picoseconds. If energy is given to a molecule to some of its vibrational, rotational, or translational degrees of freedom in the condensed phase, the molecule starts to distribute the excess energy to other degrees of freedom within the molecule. At the same time, the molecule cools down by transferring some of the energy to the surrounding molecules. By monitoring the cooling process of the molecule that receives the excess energy, we learn about the intermolecular energy transfer, which is one of the elementary processes that crucially affect a chemical reaction. Some of the Raman bands observed with picosecond TRRS are sensitive to the temperature of the molecule. A good example is the first excited singlet (S1) state of trans-stilbene. Its C]C stretch band at 1570 cm1 changes its position reflecting the temperature of the molecule. We are able to measure the cooling process of S1 trans-stilbene, photoexcited from the ground state with vibrational excess energy, by using the position of the 1570 band as a “picosecond Raman thermometer.”10 The rate constant for the cooling process observed for 10 organic solvents showed a good correlation with the thermal diffusivity of the bulk solvent. This correlation was used later for estimating the thermal diffusivity within the local structure in ionic liquids.9 Intensity ratio of anti-Stokes and Stokes Raman band reflects the population distribution among vibrational levels. Time dependence of anti-Stokes Raman bands is used as a thermometer as well. Vibrational relaxation processes of simple liquids11,12 and proteins13 are clearly observed by picosecond measurement of anti-Stokes Raman band intensities following the irradiation by an infrared or near-infrared light pulse that prepares vibrationally excited states. Fluorescence signals from the sample molecule or from its impurities may hinder a Raman measurement severely. An
Fig. 6 Schematic diagram of picosecond time-resolved Raman spectrometer.9
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Fig. 7 Optical layout for Kerr-gated Raman collection. Reprinted from Fluorescence suppression in resonance Raman spectroscopy using a high-performance picosecond Kerr gate. P. Matousek, M. Towrie, C. Ma, M. W. Kwok, D. Phillips, W. T. Toner, A. W. Parker, J. Raman Spectrosc. 32. Copyright (c) (2001) Wiley.
effective method for eliminating the fluorescence signals while recording a Raman spectrum is the use of the optical Kerr gate.14 In this method, Raman and fluorescence created from the sample by a picosecond excitation light pulse go through a pair of crossed polarizers (Fig. 7). Another picosecond light pulse acting as a “gate-pulse” irradiates a Kerr medium placed between the polarizers, inducing birefringence there. A portion of the light goes through the polarizers only when the picosecond gate-pulse induces the birefringence between the polarizers. The fluorescence intensity after the Kerr gating is reduced significantly because the most of the fluorescence signals that lasts for several nanoseconds or longer is not able to go through the crossed polarizers. The Raman signals that follow the time profile of the picosecond excitation pulse are reduced only by the Kerr gate transmittance. The Kerr gate method effectively suppresses the fluorescence background while keeping the excitation wavelength where the sample absorbs and emits fluorescence, as required for resonance Raman spectroscopy.
TRRS with Stimulated Raman Scattering Stimulated Raman spectroscopy with a time-resolving capability of pico- or subpicoseconds has been developed since the late 1990s, although the stimulated Raman spectroscopy itself has a longer history. For time-resolved stimulated Raman spectroscopy, three types of light pulses are required; the actinic pump, Raman pump, and probe pulses. The sample is irradiated by the actinic pump pulse first and is converted to a transient state. The Raman pump pulse (often called o1 pulse) and the probe pulse (o2 pulse) interact with the sample simultaneously, at a certain time delay from the actinic pump pulse, causing the stimulated Raman process. The intensity of the probe beam increases when the energy difference between the o1 and o2 pulses matches the energy spacing of vibrational levels (stimulated Raman gain). Multiplex measurement with an array detector such as a CCD is possible when white light continuum, produced with self-phase modulation of a light pulse, is used as a probe pulse.15,16 The pulse duration of the actinic pump pulse and the probe pulse is often set to be subpicoseconds, partly because it is technically easier to prepare a subpicosecond light pulse than a picosecond pulse. The Raman pump pulse, however, should be a few picoseconds or
Fig. 8 Time-resolved near-infrared stimulated Raman spectra of b-carotene in cyclohexane recorded with the Raman pump and the actinic pump wavelengths at 1190 and 403 nm, respectively. Reprinted with permission from T. Takaya, K. Iwata. Relaxation mechanism of b-carotene from S2 (1Buþ) state to S1 (2Ag) state: femtosecond timeresolved near-IR absorption and stimulated resonance Raman studies in 900-1550 nm region. J. Phys. Chem. A 118, 4071–4078. Copyright (2014) American Chemical Society.
longer because it should keep a wavenumber width of several cm1 for recording vibrational Raman spectra. The wavenumber width of a stimulated Raman band is determined by the band width imposed by the spectrometer including the width of the Raman pump pulse, and the spectral width reflecting the dephasing of the nonlinear polarization, corresponding to the free induction decay. There is no way of surpassing the timeenergy uncertainty principle even in time-resolved stimulated Raman spectroscopy using subpicosecond pulses. Use of stimulated Raman spectroscopy is effective for reducing the fluorescence background. There is no specific direction which the fluorescence light is preferably emitted to. The stimulated Raman light, however, is created by stimulated emission by the incoming probe pulse. It has the same energy, polarization, and direction as the probe pulse. If the probe beam has a specific direction of propagation, so does the stimulated
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Raman light. The intensity of the fluorescence signals is reduced substantially by placing an aperture that allows the probe beam to go through but reject most of the fluorescence light. Spontaneous Raman spectra are “background free” if there is no fluorescence background. The noise is determined by the detector. The noise of a stimulated Raman spectrum, however, is in most cases not determined by the detector but by fluctuation of the probe pulse intensity. A stimulated Raman spectrum does not show a better S/N ratio compared with the spontaneous Raman counterpart in the visible region where good detectors such as CCD are available. In the near-infrared region where the detector noise is far worse than in the visible region, however, a stimulated Raman spectrum possibly gives a better S/N ratio than spontaneous Raman. An example is shown in Fig. 8. Time-resolved near-infrared stimulated Raman spectra of b-carotene are recorded with the Raman pump wavelength of 1190 nm. Clear C]C stretch Raman bands for S2 and S1 states and their time evolution are observed at around 1550 and 1790 cm1, respectively. Stimulated Raman spectroscopy will develop as an effective method for near-infrared resonance Raman spectroscopy including TRRS.
See also: AFM and Raman Spectroscopy, Applications in Cellular Imaging and Assays; ATR and Reflectance IR Spectroscopy, Applications; Femtosecond Stimulated Raman Spectroscopy; Forensic Science, Applications of IR Spectroscopy; Forensic Science, Applications of Raman Spectroscopy to Fiber Analysis; FT-IR and Raman Spectroscopies, Polymorphism Applications; FTIR Spectroscopy of Aqueous Solutions; FT-Raman Spectroscopy, Applications; Gas Phase Raman Scattering: Methods and Applications in the Energy Industry; High Resolution Gas Phase IR Spectroscopy Applications; High Resolution Gas Phase IR Spectroscopy Instrumentation; Infrared and Raman Spectroscopy of Minerals and Inorganic Materials; IR and Raman Spectroscopies, Matrix Isolation Studies; IR and Raman Spectroscopies of Inorganic, Coordination and Organometallic Compounds; IR and Raman Spectroscopies, Polymer Applications; IR and Raman Spectroscopies, Studies of Hydrogen Bonding and Other Physicochemical Interactions; IR and Raman Spectroscopies, The Study of Art Works; IR and Raman Spectroscopy, Industrial Applications; IR Spectroscopy Sample Preparation Methods; IR Spectroscopy, Soil Analysis Applications; IR Spectroscopy, Surface
Studies; IR Spectroscopy, Theory; NIR FT-Raman; Nonlinear Raman Spectroscopy, Applications; Nonlinear Raman Spectroscopy, Instruments; Nonlinear Raman Spectroscopy, Theory; Protein Structure Analysis by CD, FTIR, and Raman Spectroscopies; Raman and Infrared Microspectroscopy; Raman Optical Activity, Applications; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Optical Activity, Small Molecule Applications; Raman Optical Activity, Spectrometers; Raman Optical Activity, Theory; Raman Spectrometers; Raman Spectroscopy, Biochemical Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Raman Spectroscopy, Soil Analysis Applications; Rayleigh Scattering and Raman Effect, Theory; Resonance Raman Applications; Spatially Offset Raman Spectroscopy; SurfaceEnhanced Raman Optical Activity (SEROA); Surface-Enhanced Raman Scattering (SERS), Applications; Surface-Enhanced Raman Scattering (SERS) Biochemical Applications; Transmission Raman: Methods and Applications; Vibrational, Rotational and Raman Spectroscopy, Historical Perspective.
References 1. See for example “Proceedings of International Conferences of Raman Spectroscopy (ICORS)” published for every two years. 2. Hamaguchi H (1987) In: Durig JR (ed.) Vibrational Spectra and Structure. Amsterdam: Elsevier Chapter 4. 3. Hamaguchi H and Gustafson TL (1994) Annu. Rev. Phys. Chem. 45: 593–622. 4. Tahara T, Hamaguchi H, and Tasumi M (1987) J. Phys. Chem. 91: 5875–5880. 5. Huang Y-S, Karashima T, Yamamoto M, and Hamaguchi H (2003) J. Raman Spectrosc. 34: 1–3. 6. Pagsberg P, Wilbrabdt R, Hansen KB, and Weisberg KV (1976) Chem. Phys. Lett. 39: 538–541. 7. Scott TW and Friedman JM (1984) J. Am. Chem. Soc. 106: 5677–5687. 8. Iwata K, Yamaguchi S, and Hamaguchi H (1993) Rev. Sci. Instrum. 64: 2140–2146. 9. Yoshida K, Iwata K, Nishiyama Y, Kimura Y, and Hamaguchi H (2012) J. Chem. Phys. 136: 104504-1–104504-8. 10. Iwata K and Hamaguchi H (1997) J. Phys. Chem. A 101: 632–637. 11. Laurbereau A and Kaiser W (1978) Rev. Mod. Phys. 50: 607–665. 12. Pein BC and Dlott DD (2013) In: Fayer MD (ed.) Ultrafast Infrared Spectroscopy, pp. 269–304. New York, NY: Taylor and Francis. 13. Mizutani Y and Kitagawa T (2002) Bull. Chem. Soc. Jpn. 75: 623–639. 14. Matousek P, Towrie M, Ma C, Kwok MW, Phillips D, Toner WT, and Parker AW (2001) J. Raman Spectrosc. 32: 983–988. 15. Yoshizawa M and Kurosawa M (2000) Phys. Rev. A 61: 013808-1–013808-6. 16. Kukura P, McCamant DW, and Mathies RA (2007) Annu. Rev. Phys. Chem. 58: 461–488. 17. Takaya T and Iwata K (2014) J. Phys. Chem. A 118: 4071–4078.
Time-resolved Raman spectroscopy (TRRS) refers to Raman spectroscopy that measures time-dependent phenomena in a time-specific manner along their time course. It possesses the ultrafast capability of laser spectroscopy and the high information content of Raman spectroscopy. TRRS has recently evolved into time- and space-resolved Raman spectroscopy (TSRRS) by the alliance with confocal optical microscopy. Highest achievable time-resolution of TRRS is subpicosecond (limited by the time-energy uncertainty principle, see below) and highest achievable space-resolution of TSRRS is a few hundred nanometers (diffraction limited). The application of TRRS/TSRRS extends tremendously in recent years not only in basic science like physics, chemistry, and biology but also in many industrial, pharmaceutical, medical, agricultural, and health sciences and technologies.1 It can measure a variety of time-dependent phenomena starting from cell division in the minute regime to ultrafast photochemical reactions in the pico- and femtosecond regimes, to provide otherwise unobtainable information based on Raman spectra (molecular fingerprints). In the early days of Raman spectroscopy (1928–60s), mercury lamps were used for Raman excitation and photographic plates were used for detection. Time-resolved measurements were not realistic since long exposure time (tens of minutes to hours) was needed for recording one Raman spectrum. After the introduction of continuous wave (CW) laser sources and photomultipliers in 1970s, sensitivity of Raman spectrometers improved markedly but the recording time was not much shortened (typically tens of minutes) because of scanning monochromators. TRRS was still not practical at this stage. Pulsed lasers and multichannel detectors introduced in 1980s changed the situation drastically. Combination of Q-switched pulsed lasers with intensified photodiode arrays (IPDA) made it feasible to perform TRRS in the micro- and nanosecond time regimes.2 Multichannel detectors facilitated the use of pulsed lasers for Raman excitation as well as Raman measurements without scanning spectrometers. Introduction of amplified mode-locked lasers advanced TRRS to the picosecond regime in 1990s and to the subpicosecond in 2000s. CCD (charge coupled device) detectors replaced IPDA after 1990s to increase the sensitivity of TRRS. The use of a narrow bandpass filter (notch filter) with a single polychromator also made the sensitivity of TRRS much higher in 2000s.3 TRRS with nonlinear Raman effects such as CARS (coherent anti-Stokes Raman scattering) and SRS (stimulated Raman scattering) was also developed after 1990s in the pico- and subpicosecond time regimes. TRRS can be classified into two categories according to time regimes that it measures. One is to look at slow changes in the millisecond or longer time regimes. RRS in this time regime is readily performed with the use of a CW laser source and a CCD detector. A commercial Raman spectrometer is capable of doing this job without much difficulty. The other is to look at changes in the ultrafast time regimes of micro-,
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
nano-, pico-, and subpicosecond. TRRS in these time regimes, ultrafast TRRS, needs pulsed laser sources for excitation and (gated) multichannel detectors for detection. Special setups specifically designed for specific purposes are necessary. Ultrafast TRRS commonly adopts the pump-probe resonance Raman scheme, in which a pump-laser pulse first photo-excites the sample to start the change and then a probe-laser pulse with varied delay times probes resonance Raman scattering from the changing sample. In this regard, TRRS is sometimes called timeresolved resonance Raman (TR3) spectroscopy. The principle of pump-probe experiment is schematically shown in Fig. 1 for nanosecond TRRS of benzophenone in the lowest excited triplet state.
TSRRS in the Hectosecond Regime An example of TSRRS application to fundamental biology is given in Fig. 2, in which a single living fission yeast (Schizosaccharomyces pombe) is measured with time-resolution of 100 s and space-resolution of 250 nm.5 It elucidates the change of molecular compositions that occurs at the central part (marked by asterisks in the figure) of the cell. Fission yeast repeats cell division according to the cycle G2 ! M ! G1/S ! G2 with a period of a few hours. The Raman spectrum of the dividing nucleus at 0 min is dominated by known Raman bands of proteins. As the mitosis proceeds M ! G1/S (6, 11, 24, and 31 min), many strong bands ascribed to phospholipids appear. In the S period (41, 62, and 69 min), the spectra are dominated by polysaccharide bands from the septum. At 72 min, the spectrum shows protein/lipid bands of the cell walls of the two daughter cells. This is the first in vivo Raman observation of the dynamic behavior of biomolecules during the cell cycle. The change indicates clearly that the cell is actually “living” during the Raman measurement.
TRRS in the Micro- and Nanosecond Regimes The first ultrafast pump-probe TRRS experiment was reported in 1976 by Pagsberg et al.6 They excited p-terphenyl by pulse radiolysis with an electron pulse (2 MeV, 30 ns duration) from a Febetron accelerator and probed the generated p-terphenyl anion radical with a flash-lamp pumped dye laser pulse at 481.6 nm (10 mJ, 600 ns). Following this work, many pumpprobe TRRS experiments were reported in the micro- and nanosecond regimes. Pulsed Q-switched lasers, Q-switched Nd:YAG lasers in particular, were most suitably used for both pumping (photoexcitation) and probing. A typical set up for nanosecond TRRS is shown in Fig. 3. As an example of TRRS in the micro- and nanosecond regimes, time-resolved Raman spectra of transient deoxyhemoglobin (human) in the time range of 10 ns to 120 ms are shown
http://dx.doi.org/10.1016/B978-0-12-409547-2.12166-3
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Fig. 1 Nanosecond pump-probe time-resolved Raman spectroscopy of the lowest excited triplet (T1) state of benzophenone.4 The fourth harmonic of a Q-switched Nd:YAG laser (266 nm) was used for pumping benzophenone from the ground (S0) to the lowest excited singlet (S1) state and the second harmonic (532 nm) in resonance with the Tn T1 electronic transition with delay time of 15 ns was used probing resonance Raman scattering from T1 benzophenone. The T1 Raman spectrum (c) is obtained as a difference spectrum between the pump þ probe spectrum (a) and the probe only spectrum (b).
Fig. 2 Time- and space-resolved Raman spectra of a dividing fission yeast cell, whose nuclei are labeled by GFP so that the positions and the number of nuclei are directly seen as fluorescence images (shown on the right-hand side). Asterisks indicate the position of the focused exciting laser beam. The 632.8 nm line of a He–Ne laser was used with 1 mW power at the sample point.
in Fig. 4.7 In this experiment, carboxyhemoglobin is photolyzed to induce the dissociation of CO from the heme and to produce transient deoxyhemoglobin. Resonance Raman spectra of this transient deoxyhemoglobin are observed in the time course of its relaxation. In Fig. 4, time evolution of
iron-proximal histidine stretch frequency is clearly observed showing the submicrosecond structure relaxation of transient deoxyhemoglobin. This experiment, together with similar TRRS experiments, has contributed significantly to the elucidation of the hemoglobin cooperativity mechanism, in which the
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Fig. 3 Block diagram of a typical nanosecond TRRS system in the 1980s.2 The second, third, and fourth harmonics of a Q-switched Nd:YAG laser and outputs from a dye laser pumped by the second harmonic were used for pumping and/or probing. A triple polychromator equipped with a subtractive double grating filter was used for a wide wavelength-range measurement. An intensified photodiode array detector (IPDA) was used for nanosecond time-gated detection to eliminate longer lifetime fluorescence/phosphorescence.
heme ligation states and the protein tertiary/quaternary structures are linked through the strain of the iron-histidine linkage of the heme.
TRRS in the Pico- and Subpicosecond Regimes
Fig. 4 Time-resolved Raman spectra of photolyzed COHb in the time range of 10 ns to 120 ms after photolysis.7 The 405 nm output (10 ns pulse duration) of a nitrogen laser pumped dye laser was used for pumping and the 435 nm output (10 ns) of an excimer laser pumped dye laser was used for probing. The pump and probe pulses were synchronized electronically with varied delay time.
Advancement of laser technology in 1990s has made it possible to produce light pulses in the ultraviolet to infrared region with pulse duration of pico- to subpicoseconds. These light pulses have been used for TRRS in the pico- and subpicosecond regimes. There is a minimum value, however, for the product of the energy width and the time width of a light pulse. A light pulse is well described as a wave packet of electromagnetic waves with different frequencies. The electric field of a light pulse as a function of frequency E(n) is converted by Fouriertransformation to the electric field as a function of time, E(t). The product of the frequency width and time width is analytically calculated for a light pulse with several pulse envelopes including the Gaussian, Lorentzian, and sech functions. These values are readily converted to the frequency–time product for the light intensity which is proportional to the square of the electric field of the light pulse. If the frequency–time product of light pulses matches the product value expected from the Fourier transform, then these pulses are called “Fourier-transform limited pulses.” The wavenumber width full width at half maximum (FWHM, cm1) of a Fourier-transform limited pulse is plotted against the duration (FWHM, ps) for Gaussian and sech2 pulses in Fig. 5.8 For a Gaussian pulse, for example, the wavenumber width is equal to or larger than 4.9 cm1 if
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the pulse duration is 3 ps. The pulse duration should be 3.0 ps or longer if wavenumber width of 5 cm1 is required for the measurement. The limitation imposed by the minimum wavenumber–time product is important when designing a TRRS measurement in a pico- or subpicosecond regime. Conventional pump-probe technique is used for picosecond time-resolved spontaneous Raman spectroscopy. Schematic diagram of a picosecond time-resolved Raman spectrometer is shown in Fig. 6.9 The pump and probe pulses with 2 ps duration for this spectrometer are prepared at two OPAs (optical parametric amplifiers) pumped by the output from a Ti:sapphire regenerative/multipass amplifier. The wavelengths of the pump and probe pulses are independently
Fig. 5 Relation between wavenumber width (FWHM, Dn) and pulse duration (FWHM, Dt) of Fourier-transform limited light pulses with pulse shape of Gaussian (a) and sech2 (b).
tunable. A 4f-filter formed by a pair of gratings and a slit is placed for limiting the energy width of the probe pulse. The intermolecular energy transfer process proceeds in picoseconds. If energy is given to a molecule to some of its vibrational, rotational, or translational degrees of freedom in the condensed phase, the molecule starts to distribute the excess energy to other degrees of freedom within the molecule. At the same time, the molecule cools down by transferring some of the energy to the surrounding molecules. By monitoring the cooling process of the molecule that receives the excess energy, we learn about the intermolecular energy transfer, which is one of the elementary processes that crucially affect a chemical reaction. Some of the Raman bands observed with picosecond TRRS are sensitive to the temperature of the molecule. A good example is the first excited singlet (S1) state of trans-stilbene. Its C]C stretch band at 1570 cm1 changes its position reflecting the temperature of the molecule. We are able to measure the cooling process of S1 trans-stilbene, photoexcited from the ground state with vibrational excess energy, by using the position of the 1570 band as a “picosecond Raman thermometer.”10 The rate constant for the cooling process observed for 10 organic solvents showed a good correlation with the thermal diffusivity of the bulk solvent. This correlation was used later for estimating the thermal diffusivity within the local structure in ionic liquids.9 Intensity ratio of anti-Stokes and Stokes Raman band reflects the population distribution among vibrational levels. Time dependence of anti-Stokes Raman bands is used as a thermometer as well. Vibrational relaxation processes of simple liquids11,12 and proteins13 are clearly observed by picosecond measurement of anti-Stokes Raman band intensities following the irradiation by an infrared or near-infrared light pulse that prepares vibrationally excited states. Fluorescence signals from the sample molecule or from its impurities may hinder a Raman measurement severely. An
Fig. 6 Schematic diagram of picosecond time-resolved Raman spectrometer.9
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Fig. 7 Optical layout for Kerr-gated Raman collection. Reprinted from Fluorescence suppression in resonance Raman spectroscopy using a high-performance picosecond Kerr gate. P. Matousek, M. Towrie, C. Ma, M. W. Kwok, D. Phillips, W. T. Toner, A. W. Parker, J. Raman Spectrosc. 32. Copyright (c) (2001) Wiley.
effective method for eliminating the fluorescence signals while recording a Raman spectrum is the use of the optical Kerr gate.14 In this method, Raman and fluorescence created from the sample by a picosecond excitation light pulse go through a pair of crossed polarizers (Fig. 7). Another picosecond light pulse acting as a “gate-pulse” irradiates a Kerr medium placed between the polarizers, inducing birefringence there. A portion of the light goes through the polarizers only when the picosecond gate-pulse induces the birefringence between the polarizers. The fluorescence intensity after the Kerr gating is reduced significantly because the most of the fluorescence signals that lasts for several nanoseconds or longer is not able to go through the crossed polarizers. The Raman signals that follow the time profile of the picosecond excitation pulse are reduced only by the Kerr gate transmittance. The Kerr gate method effectively suppresses the fluorescence background while keeping the excitation wavelength where the sample absorbs and emits fluorescence, as required for resonance Raman spectroscopy.
TRRS with Stimulated Raman Scattering Stimulated Raman spectroscopy with a time-resolving capability of pico- or subpicoseconds has been developed since the late 1990s, although the stimulated Raman spectroscopy itself has a longer history. For time-resolved stimulated Raman spectroscopy, three types of light pulses are required; the actinic pump, Raman pump, and probe pulses. The sample is irradiated by the actinic pump pulse first and is converted to a transient state. The Raman pump pulse (often called o1 pulse) and the probe pulse (o2 pulse) interact with the sample simultaneously, at a certain time delay from the actinic pump pulse, causing the stimulated Raman process. The intensity of the probe beam increases when the energy difference between the o1 and o2 pulses matches the energy spacing of vibrational levels (stimulated Raman gain). Multiplex measurement with an array detector such as a CCD is possible when white light continuum, produced with self-phase modulation of a light pulse, is used as a probe pulse.15,16 The pulse duration of the actinic pump pulse and the probe pulse is often set to be subpicoseconds, partly because it is technically easier to prepare a subpicosecond light pulse than a picosecond pulse. The Raman pump pulse, however, should be a few picoseconds or
Fig. 8 Time-resolved near-infrared stimulated Raman spectra of b-carotene in cyclohexane recorded with the Raman pump and the actinic pump wavelengths at 1190 and 403 nm, respectively. Reprinted with permission from T. Takaya, K. Iwata. Relaxation mechanism of b-carotene from S2 (1Buþ) state to S1 (2Ag) state: femtosecond timeresolved near-IR absorption and stimulated resonance Raman studies in 900-1550 nm region. J. Phys. Chem. A 118, 4071–4078. Copyright (2014) American Chemical Society.
longer because it should keep a wavenumber width of several cm1 for recording vibrational Raman spectra. The wavenumber width of a stimulated Raman band is determined by the band width imposed by the spectrometer including the width of the Raman pump pulse, and the spectral width reflecting the dephasing of the nonlinear polarization, corresponding to the free induction decay. There is no way of surpassing the timeenergy uncertainty principle even in time-resolved stimulated Raman spectroscopy using subpicosecond pulses. Use of stimulated Raman spectroscopy is effective for reducing the fluorescence background. There is no specific direction which the fluorescence light is preferably emitted to. The stimulated Raman light, however, is created by stimulated emission by the incoming probe pulse. It has the same energy, polarization, and direction as the probe pulse. If the probe beam has a specific direction of propagation, so does the stimulated
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Raman light. The intensity of the fluorescence signals is reduced substantially by placing an aperture that allows the probe beam to go through but reject most of the fluorescence light. Spontaneous Raman spectra are “background free” if there is no fluorescence background. The noise is determined by the detector. The noise of a stimulated Raman spectrum, however, is in most cases not determined by the detector but by fluctuation of the probe pulse intensity. A stimulated Raman spectrum does not show a better S/N ratio compared with the spontaneous Raman counterpart in the visible region where good detectors such as CCD are available. In the near-infrared region where the detector noise is far worse than in the visible region, however, a stimulated Raman spectrum possibly gives a better S/N ratio than spontaneous Raman. An example is shown in Fig. 8. Time-resolved near-infrared stimulated Raman spectra of b-carotene are recorded with the Raman pump wavelength of 1190 nm. Clear C]C stretch Raman bands for S2 and S1 states and their time evolution are observed at around 1550 and 1790 cm1, respectively. Stimulated Raman spectroscopy will develop as an effective method for near-infrared resonance Raman spectroscopy including TRRS.
See also: AFM and Raman Spectroscopy, Applications in Cellular Imaging and Assays; ATR and Reflectance IR Spectroscopy, Applications; Femtosecond Stimulated Raman Spectroscopy; Forensic Science, Applications of IR Spectroscopy; Forensic Science, Applications of Raman Spectroscopy to Fiber Analysis; FT-IR and Raman Spectroscopies, Polymorphism Applications; FTIR Spectroscopy of Aqueous Solutions; FT-Raman Spectroscopy, Applications; Gas Phase Raman Scattering: Methods and Applications in the Energy Industry; High Resolution Gas Phase IR Spectroscopy Applications; High Resolution Gas Phase IR Spectroscopy Instrumentation; Infrared and Raman Spectroscopy of Minerals and Inorganic Materials; IR and Raman Spectroscopies, Matrix Isolation Studies; IR and Raman Spectroscopies of Inorganic, Coordination and Organometallic Compounds; IR and Raman Spectroscopies, Polymer Applications; IR and Raman Spectroscopies, Studies of Hydrogen Bonding and Other Physicochemical Interactions; IR and Raman Spectroscopies, The Study of Art Works; IR and Raman Spectroscopy, Industrial Applications; IR Spectroscopy Sample Preparation Methods; IR Spectroscopy, Soil Analysis Applications; IR Spectroscopy, Surface
Studies; IR Spectroscopy, Theory; NIR FT-Raman; Nonlinear Raman Spectroscopy, Applications; Nonlinear Raman Spectroscopy, Instruments; Nonlinear Raman Spectroscopy, Theory; Protein Structure Analysis by CD, FTIR, and Raman Spectroscopies; Raman and Infrared Microspectroscopy; Raman Optical Activity, Applications; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Optical Activity, Small Molecule Applications; Raman Optical Activity, Spectrometers; Raman Optical Activity, Theory; Raman Spectrometers; Raman Spectroscopy, Biochemical Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Raman Spectroscopy, Soil Analysis Applications; Rayleigh Scattering and Raman Effect, Theory; Resonance Raman Applications; Spatially Offset Raman Spectroscopy; SurfaceEnhanced Raman Optical Activity (SEROA); Surface-Enhanced Raman Scattering (SERS), Applications; Surface-Enhanced Raman Scattering (SERS) Biochemical Applications; Transmission Raman: Methods and Applications; Vibrational, Rotational and Raman Spectroscopy, Historical Perspective.
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