3048
J. Phys. Chem. B 2007, 111, 3048-3054
Picosecond Optical Thermometry of Protein in H2O George A. Marcus* and H. Alan Schwettman Hansen Experimental Physics Lab, 445 Via Palou, Stanford UniVersity, Stanford, California 94305, and SUNY Geneseo, Department of Physics and Astronomy, 1 College Circle, Geneseo, New York 14454 ReceiVed: October 6, 2006; In Final Form: January 11, 2007
We demonstrate the use of transient IR absorption measurements for picosecond thermometry of protein in an aqueous environment. For small temperature changes, measured transient absorption changes are shown to be in excellent agreement with the “static” temperature dependence of the protein and water constituents of the sample as measured by FTIR spectroscopy. The thermally induced changes in IR absorption reach equilibrium within a few picoseconds, making this technique an excellent tool for picosecond thermometry.
1. Introduction Timescales and pathways of energy flow in biological systems are of considerable interest. Single-color vibrational relaxation measurements provide important information about the rate of energy flow out of specific vibrational modes that have been excited to nonequilibrium populations.1-5 Two color pumpprobe experiments can provide more information, tracking energy flow from one mode to another within a substance.6 Sometimes, however, it is important to monitor energy flow from one substance to another, as has been demonstrated by Dlott et al. to track energy flow between micelles or molecules and a solvent.7,8 This type of technique might also be useful for monitoring energy flow between different constituents in tissue or the transfer of energy from a protein to its solvent or vice-versa. The theory of using an organic solvent as such a molecular thermometer has been explored by Stratt et al.9 Measurements of this sort require that the deposited energy equilibrates rapidly between all locally accessible modes and necessitate the validation of suitable molecular thermometers. A viable molecular thermometer will have a response time comparable to the local equilibration time but it must also exhibit a spectral change that can be readily interpreted in terms of temperature. The temperature dependence of the water or deuterium association band has been used effectively as a thermometer monitoring thermalization of a biological system with its solvent.10 Lian et al.11 used this type of solvent thermometry to monitor photoexcited heme proteins thermalizing with a deuterated water solvent bath on the picosecond time scale. In a manner that is analogous to this solvent thermometer, it is possible to provide a protein thermometer based on the amide II band. Using the thermal shift in the amide II absorption mode in the protein as a secondary thermometer, we detect changes in the protein temperature for timescales longer than the equilibration time of the lineshift, which, for modest temperature changes far from the protein denaturation temperature, is a few picoseconds. Using this protein thermometer, we can study thermalization rates and thermal diffusion associated with macroscopic tissue structural elements.12 In this paper, we confirm the viability of picosecond thermometry in pure water by comparing measurements of static * Corresponding author. E-mail:
[email protected].
temperature-dependent lineshifts with transient absorption measurements. We then demonstrate picosecond thermometry of the protein component of a soft tissue sample, using the initial water thermometry data to isolate the protein response. We have selected bovine corneal stroma as a soft tissue system because it has a well characterized and regular structure which yields readily calculable and detectable temperature changes for its protein and water constituents. Different temperatures in the protein and water of the tissue sample, induced by a mid-IR heating pulse absorbed by protein and water modes in the tissue, are monitored on the picosecond time scale as they equilibrate. 2. Experimental Methods 2.1. Temperature-Dependent IR Absorption Spectra. The water and protein constituents of hydrated cornea have overlapping vibrational absorptions throughout the mid-IR. We study the temperature dependence of IR absorption in water using a pure water sample. We then examine a hydrated tissue sample, separating the water and protein contributions to the tissue absorption spectrum by identifying and subtracting the water IR absorption from the total spectrum. Although the spectral response of free water is not identical to the response of bound and hindered water molecules associated with the protein constituents of the tissue, FTIR spectra indicate that the water component of the tissue absorption spectrum is dominated by the free water that is present. 2.1.1. Water. To quantify the temperature-dependent IR absorption in water, we make careful measurements of the spectrum as a function of temperature from 25 to 55 °C. A 9 µm film of filtered, distilled and deionized water is sealed in a cell consisting of two CaF2 windows, separated by a Teflon spacer and clamped together in a copper sample mount. The temperature is regulated within 0.05 °C with a bi-directional thermo-electric cooler feeding back on a thermistor affixed to one of the cell windows. The absorption spectra are recorded as a function of temperature with a spectral resolution of 4 cm-1 using a Bruker IFS-66V FTIR spectrometer. These measurements are displayed in Figure 1 as absorption difference spectra referenced to 25 °C. The temperature dependence of these spectra is due to the influence of hydrogen bonding on the absorption line positions and linewidths. As the temperature increases, the average number and strength of the hydrogen
10.1021/jp0665832 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/01/2007
Thermometry of Protein in H2O
Figure 1. The absorption difference spectra for water, referenced to 25 °C, are displayed in the upper plot. The vertical lines indicate selected wavelengths for which the linearity of the temperature dependence of the IR absorption is evaluated. The linear responses are displayed for the bend mode in the lower left and for the association band in the lower right.
bonds diminishes. A reduction in hydrogen bonding lowers the energy barrier for hindered rotations, causing the association band (∼2000 cm-1) to shift to lower energies. Although the position of the bend mode (∼1640 cm-1) shifts very little, the line width of the mode narrows with increasing temperature. The measurements show that the absorption changes linearly with temperature on the blue side of the association band from 2150 to 2500 cm-1, as expected from the temperature-jump spectroscopy literature. In the vicinity of the bend mode, there are quasi-isobestic points at 1650 and 1590 cm-1, one on each side of the absorption peak. At the quasi-isobestic points, there is very little absorption change with temperature. These are excellent wavelength regions to use for probing thermal changes in the protein component of a tissue sample with minimal interference from the temperature dependence of the water spectrum. 2.1.2. Hydrated BoVine Corneal Stroma. We measure the IR absorption spectra of physiologically hydrated corneal stroma in a manner identical to the water measurements. At physiological hydration, bovine corneal stroma is 76% water, 23% protein and 1% lipid by mass. The majority of the protein mass (75%) is in the form of collagen,13 most of which exists as regularly ordered fibrils with uniform diameter of 38.2 ( 2 nm.14 Corneal collagen fibrils are primarily type I collagen, a rodshaped molecule that is composed of three 310-helical polypeptide chains, stabilized via internal-hydrogen bonding as well as water-bridged bonding. The fibrils are separated by an extrafibrillar matrix filled primarily with water. The protein in this tissue is quite uniform with a single dominant secondary structure, reducing inhomegeneity in the protein behavior. This tissue is extremely robust,15 making it easy to prepare thin cryosectioned samples of 10 µm thickness appropriate for IR transmission measurements. Physiologically hydrated samples of corneal stroma are created from cryo-sectioned stroma by rehydrating them with pure water and placing them in a closed chamber maintained at 100% relative humidity at room temperature. The samples are allowed to soak in the humidity chamber until IR transmission measurements show that the IR water-to-protein absorption ratio had reached the physiological level, at which time the sample cells are permanently sealed. A sealed cell consists of the sample substrate and a second CaF2 window, separated by an O-ring, clamped together in the copper sample mount. A
J. Phys. Chem. B, Vol. 111, No. 11, 2007 3049
Figure 2. The IR absorption of physiologically hydrated stroma is displayed in the spectral region encompassing the H2O bend and association modes as well as the collagen amide I and II modes. The contributions attributed to water and the hydrated collagen fibrils are displayed on the same plot.
small reservoir of water is introduced into the bottom of the sample space to ensure that physiological hydration is maintained. The sample is recharacterized at various times during the course of experiments to confirm that physiological hydration is maintained. As indicated earlier, protein and water contributions to the tissue absorption spectrum can be separately identified by subtracting the water spectrum from the hydrated cornea spectrum. As there is no protein absorption in the spectral region from 1900 cm-1 to 2500 cm-1, we can attribute the entire absorption feature in this range to the association band of water. We use an automated routine, described by Dousseau et al.,16 that subtracts a scaled water absorption spectrum from the tissue spectrum and determines the best scaling parameter. The residual spectrum, attributed to the protein, is determined with a precision of ∼2 × 10-4. The absorption spectrum of a tissue sample at 25 °C, along with the separate water and protein contributions, is displayed in Figure 2. The protein portion of the absorption spectrum exhibits spectral features that are common to all proteins, the amide I and amide II bands which are located at approximately 1650 cm-1 and 1550 cm-1 respectively. The amide I band arises predominantly from the CO stretch of the carboxyl group. The amide II band is mixed in character arising from a combination of the NH in-plane bending mode and the CN stretch mode. Careful spectral measurements were made on the tissue samples at temperatures well below 60 °C, the denaturation temperature for fibril aggregated collagen. The temperaturedependent water absorption was subtracted out, revealing the temperature dependence of the protein absorption. These absorption difference spectra are displayed in Figure 3. Both amide bands are quite sensitive to temperature induced changes in hydrogen bonding in the local environment that result from changes in both the secondary structure and the solvent. Over the measured temperature range, these lineshifts were completely reversible. As the temperature increases, the change in the amide I band absorption exhibits a complicated substructure. Hydrogen bonding with nearby amino-acid residues or solvent molecules can dramatically shift the position and shape of the CdO stretch of the amide I band, leading to an extremely sensitive dependence on minor differences in secondary structure and the presence of numerous components to the response due to inhomogeneous local environments. In contrast, the amide II mode, primarily a bend mode and backbone stretch, is less sensitive to structural hydrogen bonding changes, making it easier to interpret the spectral changes in terms of thermally
3050 J. Phys. Chem. B, Vol. 111, No. 11, 2007
Figure 3. The absorption difference spectra of the protein component of physiologically hydrated corneal stroma referenced to 20 °C. Although there is complicated structure in the amide I response, the amide II response is readily interpretable as a simple redshift in the line position.
induced changes in the local hydrogen-bonding. The amide II absorption increases on the red side of the band and decreases on the blue side of the band which is a clear signature of a simple redshift in the line position. 2.1.3. Expected Timescale for Spectral Thermalization. Having measured the temperature-dependent mid-IR absorption lines in water and protein, we examine the question of the expected time scale for thermalization of the absorption spectrum. Thermalization implies that energy deposited in the system by a heating pulse reaches some local equilibrium. For water in the mid-infrared region, there are two potentially rate-limiting steps: The lifetime of the water vibrational modes and the equilibration time scale of a non-thermal distribution in the water hydrogen-bonding network. The vibrational lifetime of the bend mode of water has been measured to be ∼1.4 ps.1 The thermalization of the hydrogen bond network has been measured in HOD:H2O following OD excitation. Though the hydrogen bond-breaking time scale is 0.8 ps, the overall hydrogen-bond equilibration time is 1.5 ps.2 Both the vibrational relaxation time and the hydrogen-bond equilibration time indicate that transient absorption changes measured in water can serve as an excellent monitor of water temperature for processes that occur on timescales longer than a few picoseconds. In protein, vibrational relaxation also represents a potential limiting factor in the thermalization of the spectrum. Vibrational lifetime measurements of the amide I band in a dry collagen film17 show a vibrational lifetime on the order of a picosecond, quite similar to measurements on the amide I band in globular proteins like Mb in solution. In Mb, this relaxation rate is only weakly dependent on temperature3 implying that the relaxation is a low-order process which does not involve numerous lowenergy modes. We must additionally consider the spatial re-distribution of energy within the protein, considering the possibility of protein conformational changes and the time scale for reaching conformational equilibrium. It is known that the amide absorption bands are sensitive to protein structure through local hydrogenbonding between amide groups. This is more pronounced in the amide I band than the amide II band as the CdO stretch of the amide I with its strong hydrogen-bonding plays a greater role in secondary structure stability than the C-N backbone stretch and the in-plane C-N-H bend of the amide II band. At the denaturation threshold, large scale unfolding of secondary structure elements can occur on a time scale ranging from 10 ns to hundreds of microseconds.18 However, in the limit of small temperature changes far from the denaturation temperature, there
Marcus and Schwettman
Figure 4. General schematic of 2-color mid-IR pump-probe apparatus.
are no substantial global conformational changes. In a measurement with instrument limited temporal response, the thermalized spectrum in a transiently heated globular protein has been observed on a time scale faster than 35 ps.19 Our experiments focus on absorption changes in the amide II band due to small changes in temperature, at temperatures far from denaturation. Under these conditions, the influence of conformational change on the protein response is limited and we can expect the protein response to be rapid. 2.2. Picosecond Thermometry. 2.2.1. Experimental System. We demonstrate picosecond protein thermometry using a twocolor mid-IR pump mid-IR probe spectroscopy technique. A pump laser generates a heating pulse, inducing a temperature change in the tissue sample. The resulting changes in the protein and water temperatures are detected with a separate probe laser that measures the transient absorption changes to detect the thermalization between the tissue constituents. A schematic layout of the experiment appears in Figure 4. A picosecond optical parametric amplifier (Spectra-Physics OPA-800) equipped with a difference frequency crystal generates ∼3 µJ pulses in the mid-infrared tunable from 3333 to 1000 cm-1 (3 to 10 µm). This source is used as a pump to rapidly heat the sample by targeting specific protein and/or water absorptions. The pump-induced changes in the sample absorption are measured at a probe wavelength generated by the Stanford free electron laser (FEL). The probe laser delivers an 11.8 MHz train of 1 µJ pulses at a pulsed 20 Hz rate with a wavelength tunable from 2220 to 1000 cm-1 (4.5 to 10 µm). An acousto-optic modulator is used to select a “mini-bunch” of a few probe pulses for each pump pulse. A pre-pump reference pulse and a post-pump probe pulse pass through the same region of the sample, allowing us to use intensity referencing to reduce the influence of 20 Hz amplitude fluctuations on the absorption measurement. Since both the reference and probe pulse are part of the FEL pulse train, they experience identical optical transport and we can detect both pulses on a single detector with a sufficiently fast temporal response. The pulse-to-pulse amplitude fluctuations in the probe beam are suppressed by a factor of the FEL cavity Q (∼30). The two lasers in the pump-probe system are electronically synchronized resulting in an instrument impulse response time of 1.5 to 2.5 ps, as measured by cross-correlation in an AgGaSe2 crystal. A real-time diagnostic of the relative timing of the pump and probe pulses, using two-photon absorption to generate photoinduced absorption in InAs, allows us to monitor the timing drift and jitter between these lasers while experimental data is acquired.
Thermometry of Protein in H2O The OPA pump beam is collimated in a reflective telescope and directed to a computer controlled optical delay line with a maximum delay of 1.3 ns. The pump and probe beams are both focused by a single 90° off-axis paraboloid onto the sample resulting in typical spot sizes of 60 and 50 µm and energies of 1 µJ and 10 nJ for the pump and probes, respectively. The probe pulses are sent through a spectrometer which spectrally rejects scattered pump light. The probes are detected by an LN cooled mercury-cadmium-telluride (MCT) detector (Kolmar Technologies) with a bandwidth of 50 MHz which is sufficient to temporally resolve adjacent micropulses. An RF splitter divides the detector signal between two independent gated integrators, one of which integrates the reference pulse while the other integrates the signal pulse. The output of the gated integrators is digitized, recorded and analyzed by a PC. The natural log of the ratio (transmitted probe/ transmitted reference) is multiplied by -1 to yield a measure of the pump induced change in absorbance. Data is collected as a function of delay time between the pump and the probe pulses. The entire optical apparatus is contained in a purge chamber to eliminate interference from atmospheric IR absorption lines. The transient absorption measurements are fit to a simple exponential equilibration model that is convolved with a measured instrument response function. The model contains a fast and slow component to the response. The signal, S(t), should be Ainit(1 - e(t-t0)/τfast) +(Aeq - Ainit)e(t-t0)/τeq + C. The fast component amplitude, Ainit, corresponds to the absorption change associated with the initial temperature jump. The slow component corresponds to the subsequent thermalization process as the temperatures of the multiple components of the sample equilibrate and the response relaxes to the equilibrated absorption response, Aeq, with an equilibration time, τeq. In a single component sample, like pure water, the model reduces to the first term in the expression above. 2.2.2. Predicting the Amplitude of Induced Absorption Changes. To demonstrate that the transient absorption response is a meaningful measurement of temperature, it is important to show that the amplitude of the transient absorption change corresponds to the absorption changes expected from the FTIR measurements of temperature-dependent IR spectra. To accomplish this, we must determine the amplitude of the temperature jump generated by the IR heating pulse and then calculate the expected change in the probe absorption, based on the FTIR temperature-dependent absorption spectra. This type of calculation has been described in detail by Dlott et al.7 In short, the spatial distribution of the pump induced temperature change is determined by the deposited energy density in the heated sample. The calculation in this experiment is slightly more complicated in that the sample has multiple constituents with different properties. The penetration depth of the heating pulse is determined by the average optical properties of the sample. However, each constituent in the sample region will initially be at a different temperature, depending upon the fraction of the deposited energy absorbed and the thermal properties of the constituent. The average temperature seen by the probe beam is determined by properly accounting for the different contributions of each constituent, due to temperature changes, temperature-dependent spectral changes, and penetration depths. After thermalization has occurred, the temperature of all constituents will be the same, as predicted by the average properties of the sample. Implicitly, a calculation such as this assumes that the change in absorption in each constituent is linearly proportional to the change in the temperature. We limit our experiments to
J. Phys. Chem. B, Vol. 111, No. 11, 2007 3051 TABLE 1: Temperature Jumps and Absorption Changes in Sample of Physiologically Hydrated Corneal Stroma Following a Heating Pulse Tuned to 1680 cm-1 under Typical Experimental Conditions sample constituent
init. temp. jump
init. ∆R (µm-1 )
eq. temp. jump
eq. ∆R (m-1)
protein water
Probing the Response at 2170 cm-1 6.8 K 0.0 × 10-4 2.5 K 2.0 K -4.0 × 10-4 2.5 K
0.0 × 10-4 -5.0 × 10-4
protein water
Probing the Response at 1570 cm-1 6.9 K -7.7 × 10-4 2.6 K 2.1 K -1.3 × 10-4 2.6 K
-4.2 × 10-4 -1.6 × 10-4
temperature ranges and changes in which this approximation is valid by inducing small temperature rises and ensuring that no sample constituents undergo dramatic alterations such as phase changes (boiling in water) or structural changes (large-scale unfolding or denaturation in protein). Assuming typical experimental conditions, as described in the previous section, with the heating pulse tuned to 1680 cm-1 simultaneously exciting the amide I and water bend modes, the predicted temperature and absorption changes in a sample of physiologically hydrated corneal stroma are presented in Table 1. The initial temperature describes the differentially heated tissue immediately following the heating pulse. The equilibrium temperature is reached some time later after the differentially heated protein and water constituents thermalize with each other. There are a few important elements highlighted by these predicted responses. The protein temperature changes much more dramatically than the water temperature. This is due to the smaller thermal mass of the protein. This larger swing in protein temperature makes it easier to detect the difference between the initial and equilibrium absorption due to the protein than it is to detect the changes in the water component of the absorption. In this example, if we probe the changes in absorption at 1570 cm-1, where the response is dominated by the protein temperature, the difference between the initial and equilibrium absorbance is 3.2 × 10-4 per µm. However, if we probe at 2170 cm-1 where the response is dominated by the water temperature, the difference between the initial and equilibrium absorbance is only 1.0 × 10-4 per µm. A second thing to note is that the average expected temperature changes are slightly different at the two probe wavelengths. This is due to the mismatch between the penetration depth of the probe beam and the heating pulse at the different wavelengths. In the case of the probe at 2170 cm-1, the heating pulse deposits energy in a shallower region than the volume sampled by the probe, leading to smaller average temperature changes. 3. Results and Discussion 3.1. Water. Transient absorption changes were measured at various probe wavelengths in the bend mode from 1625 to 1540 cm-1. The pump pulse was tuned to the blue side of the bend at 1710 cm-1. A typical transient absorption measurement in the bend mode of pure water is displayed in Figure 5. It is clear that the transient absorption change has equilibrated within a few picoseconds. The equilibrated absorption change was also measured out to 1 ns and remains constant within experimental resolution. Since this change is in agreement with the FTIR temperature difference spectra, we can conclude that we are measuring thermally induced spectral changes within a few picoseconds. At each probe wavelength, the measured absorption change is scaled by the calculated temperature jump to determine the absorption change per degree. As the upper panel of Figure 6
3052 J. Phys. Chem. B, Vol. 111, No. 11, 2007
Figure 5. Transient absorption in the water bend mode. The induced temperature jump was 2.2 °C. The extracted fit parameters yield a transient absorption change of 2.15 × 10-2 and an equilibration time of 3.46 ( 1.73 ps.
Marcus and Schwettman
Figure 7. Wavelength dependence of the equilibration time of the association band transient response.
Figure 8. Transient absorption in the protein amide II mode of dehydrated corneal stroma.
Figure 6. The measured wavelength dependence of the amplitude of the ultrafast transient absorption changes in the bend mode (upper panel) and the association band (lower panel) show excellent agreement with the changes expected from the FTIR measurements of the thermal lineshift.
shows, the scaled transient absorption amplitude is in excellent agreement with the response predicted from the FTIR data. Transient absorption measurements were also made for different pump wavelengths within the bend mode including the red side of the bend at 1626 cm-1 and at 1550 cm-1. These pump wavelengths lie on the red side of the probe wavelengths. There was no observable difference in the equilibration time for all pump and probe wavelengths. Transient absorption measurements in pure water made at probe wavelengths in the association band from 2030 to 2180 cm-1 also show complete equilibration during the initial picosecond transient. The lower panel in Figure 6 shows good agreement between the amplitude of the transient response and the association band response predicted from the FTIR data. As in the bend mode, this indicates that the transient absorption measurement serves as a thermometer for timescales longer than a few picoseconds. However, unlike the bend mode response, the equilibration time appears to change with wavelength, as
we see in Figure 7, indicating that we may be seeing the effects of a multistep energy relaxation pathway and/or hydrogen-bond thermalization. At the shorter wavelengths, near 2180 cm-1, we measure an equilibration time of 1.7 ( 1.2 ps. Our temporal resolution of the equilibration times is not sufficient for further exploration of this effect; Although this measured time is slightly longer than what has been reported elsewhere, it is consistent with the temporal resolution of this experiment and feedback effects associated with the “long” excitation pulses.20 The wavelength dependence of the amplitude of transient IR absorption changes in both the bend mode and the association band are in good agreement with the predictions from the temperature dependent measurements of the water IR spectrum. We conclude that the entirety of the transient response can be attributed to thermalization of the vibrational excitation into water vibrational modes and the hydrogen-bonding network on the picosecond time scale. 3.2. Dehydrated Corneal Stroma. Vibrational relaxation measurements of the amide I vibrational mode in a dehydrated protein film reveal a picosecond relaxation time.17 By making transient absorption measurements on a dehydrated corneal stroma sample (a dry collagen film), we show that the thermal lineshift of the protein absorption feature also occurs on the picosecond time scale. The sample was heated with a pump pulse tuned to the blue side of the amide I mode at 1681 cm-1 while the protein response was probed at 1570 cm-1 on the blue side of the amide II mode. The data, displayed in Figure 8, clearly shows that an equilibrium is reached within a few picoseconds. Once again, the equilibrated absorption change in the protein remains unchanged within experimental resolution out to 1 ns.
Thermometry of Protein in H2O
Figure 9. Thermalization of the protein in hydrated corneal stroma following heating pulse at 1680 cm-1. The three horizontal lines indicate the change in protein absorption (a) before the arrival of the heating pulse, (b) immediately after the heating pulse, and (c) after the deposited energy has thermalized between the protein and water constituents of the sample as determined from the fitting parameters from the exponential model of the response.
Transient absorption measurements at 1530 cm-1 on the red side of the amide II mode also showed an ultrafast response but of the opposite sign, as expected from the thermal shift of the amide II band toward the red. These results lend credence to the expectation that the protein lineshift thermalizes on the picosecond time scale. While it is difficult to accurately predict the expected magnitude of the absorption response due to large sample inhomogeneities in the dehydrated film samples, quantifiable agreement can be achieved in hydrated samples. 3.3. Hydrated Corneal Stroma. As an example of the utility of this technique for studying proteins in an aqueous environment, we examine a hydrated corneal tissue sample. Physiologically hydrated corneal stroma samples were prepared and the transient absorption was monitored at the water quasiisobestic point at 1590 cm-1 and at 1570 cm-1 on the blue side of the amide II band following an IR heating pulse. At the pump wavelength of 1681 cm-1, there is a good match between the penetration depth of the tissue and the thickness of the sample. At this wavelength, pump energy is directly deposited into both the H2O bend mode and the protein amide I mode. Much like in the example described in Table 1, the protein temperature should initially rise to ∼6.7 °C before equilibrating with the water in the tissue at a temperature of ∼2.6 °C. We measure the amplitude of the initial and equilibrium absorption response and compare these measurements to the predicted response. Using probe wavelength of 1590 cm-1 where there is no response due to the water, we measure a rapid transient absorption change with an amplitude that is in agreement with the expected temperature jump. However, at this probe wavelength the difference between the initial and equilibrium absorption change is too small for us to detect with our apparatus. As can be seen in Figure 3, the magnitude of the protein response, -1.12 × 10-4 µm-1 K-1, is 3x larger at 1570 cm-1 than at 1590 cm-1. However, at this wavelength, we must make sure to include the contribution of the water to the absorption response. Taking all factors into account, the total expected initial and equilibrium absorption responses are -7.2 × 10-3 and -4.9 × 10-3, respectively, with -2.0 × 10-3 attributable to the water. The measured transient absorption response, shown in Figure 9, yields initial and equilibrium amplitudes of -8.9 (+1.3/-2.6) × 10-3 and (-4.6 ( 1.8) × 10-3 for the absorption response, which are in excellent agreement with the predicted values within experimental error. As the data in Figure 9 shows, the protein temperature relaxes from its initial value to its equilibrium value with a time constant
J. Phys. Chem. B, Vol. 111, No. 11, 2007 3053 of ∼150 ps. The thermalization process at work in this hydrated tissue sample is examined in greater detail in a separate paper.12 One can make a complementary measurement in the corneal stroma by the measuring the transient absorption response of the water in the tissue. However, the optical and thermodynamic properties of the water result in a transient absorption change between the initial and final temperatures that is 10 times smaller than the protein response. This is very difficult to detect and is a clear indicator that protein thermometry is well suited to making measurements that may not be possible simply by monitoring the behavior of the water solvent. 4. Summary. We have developed and demonstrated a technique for picosecond thermometry of protein. This molecular thermometer makes use of the thermal lineshift of the protein amide II mode and is potentially useful for a range of biochemical studies. In particular, it is able to detect the rate of energy exchange between a transiently excited protein and the surrounding solvent (or vice-versa). This technique allows us measure the ultimate thermalization rate in biological materials which can help to identify the presence of bottlenecks in the thermalization process. This type of measurement is relevant, for example, to the study of ultrafast mid-IR laser-tissue interaction which is important in understanding ablation in structured tissue. As we have shown, an advantage of monitoring the protein temperature instead of the water temperature is that the thermally induced change in protein absorption can be much larger than the complementary change in solvent absorption. This increased sensitivity to protein temperature arises from the fact that the absorption cross section, the temperature-induced lineshift and the induced temperature jump in the protein are all large. However, the water bend mode partially overlaps with the amide II mode, making it essential to carefully characterize the thermal response of the solvent to properly isolate and interpret the protein response. For molecular systems with little spectral overlap with the water bend mode, we have demonstrated that spectral changes in the bend mode can be used for picosecond water thermometry. As the temperature dependence of the bend mode is much larger than that of the more commonly used association band, it might be used for thermometry in selected molecular systems when the temperature changes are more difficult to detect. Acknowledgment. This work was supported by the Air Force Office of Scientific Research, grant # FA9550-04-1-0075. We also acknowledge the support of the staff of the Stanford Free Electron Laser Center. References and Notes (1) Wang, Z.; Pakoulev, A.; Pang, Y.; Dlott, D. D. Chem. Phys. Lett. 2003, 378, 281-288. (2) Steinel, T.; Asbury, J. B.; Zheng, J.; Fayer, M. D. J. Phys. Chem. A 2004, 108, 10957-10964. (3) Peterson, K. A.; Rella, C.; Engholm, J. R.; Schwettman, H. A. J. Phys. Chem. B 1999, 103 (3), 557-561. (4) Zanni, M. T.; Asplund, M. C.; Hochstrasser, R. M. J. Chem. Phys. 2001, 114, 4579-4590. (5) Austin, R. H.; Xie, A.; van der Meer, L.; Redlich, B.; Lindgard, P. A.; Frauenfelder, H.; Fu, D. Phys. ReV. Lett. 2005, 94, 128101/1-128102/ 4. (6) Ashihira, S.; Huse, N.; Espagne, A.; Nibbering, E. T. J; Elsaesser, T. Chem. Phys. Lett. 2006, 424, 66-70. (7) Deak, J. C.; Iwaki, L. K.; Rhea, S. T.; Dlott, D. D. J. Raman Spectrosc. 2000, 31, 263-274. (8) Deak, J. C.; Pang, Y.; Sechler, T. D.; Wang, Z.; Dlott, D. D. Science 2004, 306, 473-476. (9) Graham, P. B.; Matus, K. J. M.; Stratt, R. M. J. Chem. Phys. 2004, 121, 5348-5355.
3054 J. Phys. Chem. B, Vol. 111, No. 11, 2007 (10) Dyer, R. B.; Gai, F.; Woodruff, W. H.; Gilmanshin, R.; Callendar, R. H. Acc. Chem. Res. 1998, 31, 709-716. (11) Lian, T.; Locke, B.; Kholodenko, Y.; Hochstrasser, R. M. J. Phys. Chem. 1994, 98, 11648-11656. (12) Marcus, G. A.; Schwettman, H. A. Manuscript in preparation. (13) Fatt, I.; Weissman, B. A. Physiology of the Eye: An Introduction to the VegetatiVe Functions; Butterworh-Heinemann: Boston, 1992. (14) Meek, K. M.; Quantock, A. J. Prog. Retinal Eye Res.h 2001, 20 (1), 95-137. (15) Meek, K. M.; Boote, C. Exp. Eye Res. 2004, 78, 503-512.
Marcus and Schwettman (16) Dousseau, F.; Therrien, M.; Pezolet, M. Appl. Spectrosc. 1989, 43, 538-542. (17) Peterson, K. A. Private communication. (18) Callendar, R. H.; Dyer, R. B.; Gilmanshin, R.; Woodruff, W. H. Ann. ReV. Phys. Chem. 1998, 49, 172-202. (19) Phillips, C. M.; Mizutani, Y.; Hochstrasser, R. M. Proc. Nat. Acad. Sci. 1995, 92, 7292-7296. (20) Bakker, H. J.; Lock, A. J.; Madsen, D. Chem. Phys. Lett. 2004, 384, 236-241.
