Bulletin of the American Physical Society, Mar 18, 2013
Florida-It is well known that the generalized Kramers-Kronig relationship is able to retrieve the... more Florida-It is well known that the generalized Kramers-Kronig relationship is able to retrieve the phase of a signal from measured power spectra. This phase recovery is a critical procedure in nonlinear optical spectroscopy, e.g. coherent Raman time domain or frequency domain spectroscopy. Several other methods have been developed and being used in the past: notably, nonlinear fitting and maximum entropy method. A firm mathematical comparison of the methods including the effects of final signal sampling and their merit of fidelity will be presented. Attention is given to numerical implementation of the phase retrieval procedure to put it into practice in coherent anti-Stokes Raman microscopy. Phase retrieval examples using all the above methods are taken from earlier and recently recorded spectra. 1 Financial support from the University of West Florida is acknowledged.
Vibrational spectra from ground-state bacteriorhodopsin (BR) in light-adapted protonated and perd... more Vibrational spectra from ground-state bacteriorhodopsin (BR) in light-adapted protonated and perdeuterated (D20) water samples (i.e. from BR-570) are recorded by picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Coherent excitation by pairs of 6 ps pulses at o, (578 nm, Aw<4 cm-') and at w, (604-643 nm, A~=300 cm-') simultaneously generates PR/CARS signals over = 300 cm-'. The high S/N of the PR/CARS spectra obtained over the 760-1750 cm-' region merit a quantitative xc3' analysis to obtain band origin positions, band shapes, relative band intensities, and phase factors describing the electronic resonance. Excitation conditions are selected to minimize BR photochemistry and ensure the electronic resonance of o, with the retinal chromophore and therefore, these PR/CARS data can be assigned to the all-trans, 15-anti retinal structure found in BR-570. Changes not only in the retinal structure itself, but also in the surrounding environment (protein and water molecules) which either alter the retinal structure or influence the electronic resonance in the retinal chromophore are reflected in these results. Thus, a more detailed view than previously available can be obtained of the vibrational degrees of freedom of all-trans, 15-anti retinal. Relative to resonance Raman data from BR-570, 8 new vibrational features in the PR/ CARS spectra from BR-570 and changes in the positions of Raman bands directly attributable to the deuteration of the Schiff base nitrogen are found. Attention also is given to the influence of non-resonant and resonant contributions to the PR/CARS signal. These results are especially important in the analysis of PR/CARS data from samples in which mixtures of BR retinal chromophores, both static and transient, are found. * Corresponding author. membrane has been shown to involve structural (configurational and conformational) transformations in both the retinal chromophore and in its surrounding protein environment [ 21. The photolytically initiated changes in the electronic absorption maxima assigned to the retinal chromophore have been used to identify two photophysical (H and I) and at least six photochemical (J, K, L, M, N and 0) intermediates which define the BR photocycle [ 31. After several milliseconds, the initial ground-state species, BR-570, is reformed from 0. The fluorescence spectroscopy of
ABSTRACT The molecular mechanism describing the initial 200 ps of the room-temperature photocycle... more ABSTRACT The molecular mechanism describing the initial 200 ps of the room-temperature photocycle in the artificial bacteriorhodopsin (BR) pigment, BR6.9, is examined by both absorption and vibrational spectroscopy. The BR6.9 pigment contains a structurally modified retinal chromophore (retinal 6.9) having a six-membered carbon ring bridging the C9C10−C11 bonds. Picosecond transient absorption (PTA) data show that the initial 200-ps interval of the BR6.9 photocycle contains two intermediates: J6.9 formed with a &lt;3-ps time constant and decaying to K6.9 with a 5-ps time constant (K6.9 has a &gt;5-ns lifetime). Resonantly enhanced vibrational spectra from the light- and dark-adapted ground states of BR6.9 are measured using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Each of these PR/CARS spectra (800−1700 cm-1) contains 33 features assignable to the vibrational degrees of freedom in the retinal chromophore. CARS spectra from the K6.9, obtained from picosecond time-resolved CARS (PTR/CARS) data using 10-ps, 50-ps, 100-ps, and 200-ps time delays following the 570-nm initiation of the BR6.9 photocycle, contain a comparable number of features assignable to the retinal in K6.9. The vibrational spectrum of J6.9 can be tentatively characterized by two bands observed in the 1120−1200-cm-1 region from the analysis of the 10-ps PTR/CARS data. Comparisons involving these PTA and CARS data from BR6.9, as well as analogous results obtained from the ground states and photocycle intermediates of native BR and other artificial BR pigments, demonstrate that restricting retinal motion at the C9C10−C11 bonds does not generally change the initial 200-ps photocycle mechanism, but does alter the rates at which specific molecular processes occur. These vibrational CARS spectra show that the retinal structures in K6.9 and in both light- and dark-adapted BR6.9 are all distinct. However, the specific mechanistic role, if any, of C13C14 isomerization cannot be directly identified from CARS data recorded from BR6.9 and its photocycle intermediates. Even though C13C14 isomerization has been widely considered the primary retinal structural change underlying the proton-pumping mechanism in BR pigments, these results leave open the question of whether C13C14 isomerization is required as a mechanistic precursor for biochemical activity in BR pigments such as BR6.9.
In the final stages of the production process, after the paper had been published, an unfortunate... more In the final stages of the production process, after the paper had been published, an unfortunate error was found in the title: anti-Raman should have been anti-Stokes Raman. Also, the seventh line of the Abstract (633.4 nm) and to s (675-700 nm) should have been (663 nm) and tos (698-745 nm).
Bulletin of the American Physical Society, Jun 1, 2018
Florida-We report the recent progress of construction and characterization of a coherent Raman ta... more Florida-We report the recent progress of construction and characterization of a coherent Raman tabletop system utilizing a novel broadband nanosecond optical parametric oscillator and volumetric Bragg filters assisting a PS-3CBCRS measuring system. To illustrate the versatility of the system and precision of the measurements to reveal the molecular information, we selected two chemicals. Polarization sensitive epi-detected 3CBCRS spectra of liquid CCl 4 and DCVJ were recorded and analyzed. By changing the polarization angles of the actinic laser waves, the vibrational bands resembled the Lorentzian line-shapes extracted from dispersive spectra by non-model dependent spectral processing methods. Electronic resonance enhanced PS-3CBCRS spectra of DCVJ was recorded for the first time. The new outcomes are additions to our recently reported 3CBCRS results on liquid and crystal samples.
Bulletin of the American Physical Society, Mar 23, 2011
The liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was in... more The liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was injected into a commercially available liquid crystal cell (INSTEC, Inc). The cell was housed in a temperature controlled environment constructed in the lab and exposed to light from a frequency doubled Nd: YAG laser. The spectrum of fluorescence from the sample was measured at several temperatures over the range 250 to 450C, covering the smectic-nematic and nematic-isotropic phase transitions. The sample was held at each temperature with a precision and resolution of 1mK before taking the spectrum. It was therefore possible to approach very close to the phase transitions. The results show a significant change in the fluorescence spectrum near the nematic-isotropic phase transition.
Bulletin of the American Physical Society, Mar 3, 2015
Florida-Laser Induced Breakdown Spectroscopy (LIBS) is a very practical spectroscopy to determine... more Florida-Laser Induced Breakdown Spectroscopy (LIBS) is a very practical spectroscopy to determine the chemical composition of materials. Recent technical developments resulted in equipment used on the MARS Rover by NASA. It is capable of measuring the emission spectra of laser induced plasma created by energetic laser pulses focused on the sample (rocks, metals, etc.). We have develop a Laser Induced Breakdown Spectroscopy setup and investigated the necessary experimental and methodological challenges needed to make such material identification measurements. 355 and 532 nm laser pulses with 5 ns temporal duration was used to generate micro-plasma from which compositions can be determined based on known elemental and molecular emission intensities and wavelengths. The performance of LIBS depends on several parameters including laser wavelength, pulse energy, pulse duration, time interval of observation, geometrical configuration of collecting optics, and the properties of ambient medium. Spectra recorded from alloys (e.g. US penny coin) and pure metals will be presented.
Spectral fidelity of any coherent Raman spectra is a major concern of building and/or using an in... more Spectral fidelity of any coherent Raman spectra is a major concern of building and/or using an instrument in "real-time" applications such as e.g. chemical component monitoring in a reaction chamber, or mapping the distribution of certain chemicals in a solid sample matrix. Mainly, three methods are used to recover the vibrational information from the measured spectra: 1. Nonlinear fitting based upon the Levenberg-Marquardt algorithm[1], 2. Maximum Entropy Method[2], and 3. Direct phase retrieval procedure based upon the assumption that the third order susceptibility is an analytic function[3]. Corrections for instrumental response and the problem of finite sampling with appropriate "windowing" are also necessary to recover the vibrational bands needed to identify or characterize the samples. These mathematical methods will be explained and a computer program written in LabView will be presented. Spectra taken by a nanosecond (at UWF)[4] and a picosecond (at UofA)[5] CARS spectrometer will be presented and analyzed by all three methods, vice infra. It is concluded that the third method is the most appropriate one for "real-time" data processing.
relevant to the interpretation of experimental spectra will be presented. The method is applied t... more relevant to the interpretation of experimental spectra will be presented. The method is applied to measure the vibrational manifold of DCVJ for the first time. DCVJ is a charge transfer molecular rotor showing a viscosity dependent fluorescence quantum yield. Based upon the measured CARS spectra, the effect of inhibition of the internal rotation on the vibrational motion of the molecule will be discussed. The design and operation of an all solid-state broadband nanosecond CARS system will be also presented. An overview of applications of molecular rotors in biology and information technology will be outlined.
Bulletin of the American Physical Society, Mar 23, 2011
liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was inject... more liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was injected into a commercially available liquid crystal cell (INSTEC, Inc). The cell was housed in a temperature controlled environment constructed in the lab and exposed to light from a frequency doubled Nd: YAG laser. Fluorescence from the sample was filtered from the incident light and detected using a photodiode and measured with a lock-in amplifier. Measurements have been made over the temperature range 25 • to 45 • C. The sample was stabilized at each temperature, and the fluorescence intensity was measured at several temperatures. The results show a significant change in fluorescence near the nematic-isotropic phase transition. The temperature control and precision allowed more than 1000 data points to be taken between 25-50 • C, with most of these clustered in the transition region between 38.5-39.5 • C, where the change in intensity was observed.
XXII INTERNATIONAL CONFERENCE ON RAMAN SPECTROSCOPY, 2010
The emerging field of nanotechnology requires new methodologies to be developed in order to addre... more The emerging field of nanotechnology requires new methodologies to be developed in order to address the needs of fabrication, manipulation, control, and measurement of motions of molecules and devices on the nanoscale. Optical excitation and control of translational, vibrational, and rotational motions can offer a unique way to achieve the desired goals by using a sequence of short laser pulses, e.g., tuned to initiate certain nuclear motions of well studied photo active proteins (e.g. PYP or Rhodopsin) or engineered larger molecular structures. Measuring nuclear motions requires structurally sensitive spectroscopy methods, and therefore spontaneous Raman and coherent Raman spectroscopies can be applied to qualitatively test and quantitatively measure such motions. Polarization Sensitive Resonance Coherent anti-Stokes Raman Spectroscopy (PSCARS), steady state or time-resolved, can measure controlled dipole moment reorientation through the anisotropic third order nonlinear optical susceptibility. The measured vibrational spectra are sensitive to photo induced isomerization or internal molecular rotation if the rotating chemical group is coupled to or interacting with internal normal modes or to the surrounding molecules e.g. in solutions. In our study, the method was applied to measure the vibrational manifold and reorientation of DCVJ for the first time. DCVJ is a charge transfer molecular rotor showing a viscosity dependent fluorescence quantum yield. This dependence is attributed to the ``friction'' type interaction between the 90 degree internal rotation of the molecule upon photo-excitation and the solvent cage surrounding it. Quantum chemical studies using DFT and normal mode calculations support our findings related to the conformational evolution of the molecule. Theoretical and experimental principles associated with CARS with an emphasis on points relevant to the interpretation of the results will be presented. The design and operation of an all solid-state broadband nanosecond CARS system will be also presented. Finally, an overview of applications of molecular rotors in biology, nanotechnology and perhaps information technology will be outlined.
Bulletin of the American Physical Society, Mar 15, 2010
capacitance measurement has been performed near the phase transitions of scientifically important... more capacitance measurement has been performed near the phase transitions of scientifically important liquid crystals such as 8-CB. The details of the measurements to get high precision data on dielectric constant and its temperature dependence will be presented. The results show significant changes of the dielectric properties of the liquid crystal near the smectic-to-nematic and nematic-to-liquid phase transitions attributed to structural changes of the relevant phases. In order to measure the details of the functional dependence near the phase transition, the temperature was varied with milliKelvin precision. The data was obtained using a self-assembled RC circuit with phase sensitive lock-in amplifier detection. Calibration of the device was made by measuring known standard capacitances. In order to get high accuracy the measurement was completely computer controlled. The Method applied here will contribute to the better understanding of thermodynamic behavior of liquid crystals and can be routinely used to characterize novel materials showing phase transitions.
Vibrational spectra recorded by coherent antiatokes resonance Raman scattering (CARS) from bacter... more Vibrational spectra recorded by coherent antiatokes resonance Raman scattering (CARS) from bacteriorhodopsin (BR) samples containing isotopically substituted (' H and 13C) retinal chromophores were measured using high repetition rate, low-power, picosecond pulsed excitation (1, = 580 nm and 1, = 640 f 3 nm). These picosecond resonance CARS (PR/CARS) data were analyzed via third-order susceptibility relationships [ ~(~' 1 to obtain band origins, bandwidths, relative intensities, and electronic phase factors assignable to all sipilkant vibrational Raman features in the 1490-1700 cm-' wavenumber region (the ethylenic stretching and C=N-H rocking or Scbiff base modes). Isotopic substitution selectively places ' H at C,, , 13C singly at the C,, position and at the C,, position, and "C simultaneously in positions of C,, and C,,. Each isotopic BR sample was examined not only in H'O, but also in D,O, which places a ' H at the Schiff base nitrogen of the retinal. In addition, PR/CARS data were recorded from each isotopic BR sample following either light adaptation lie. the BR sample contained a single retinal isomer (all-trams, 1 k t i or BR-570)I or dark adaptation 1i.e. the BR sample contained a mixture of comparable amounts of retinal isomers (BR-570 and 13-cis, 15-syn or BR-548)]. Excellent agreement was found between the vibrational features observed by PR/CARS and those obtained from spontaneous resonance Raman measurements from the same isotopically substituted BR pigments. Several new vibrational features were also found from the PR/CARS data. Vibrational Raman data from three of the isotopic BR samples in D,O are reported for the first time.
The dynamics associated with the formation bathorhodopsin (batho) intermediate in the room-temper... more The dynamics associated with the formation bathorhodopsin (batho) intermediate in the room-temperature rhodopsin (RhRT) photosequence are investigated with picosecond transient absorption (PTA) spectroscopy. The transient batho concentration is continuously monitored via absorbance changes with 2-ps time resolution for time delays as long as 500 ps. By resolving both the (5 f 1)-ps formation time constant of batho from photorhodopsin and measuring a constant batho concentration over the 20-500-ps interval, these PTA data bridge earlier studies which measured each of these processes separately but not both simultaneously. The FTA data presented here are compared both with other time-resolved absorption studies from which contradictory kinetic results have been obtained and with recently reported picosecond time-resolved vibrational studies (coherent anti-Stokes Raman) of room-temperature batho.
The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of... more The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of the E46Q mutant of photoactive yellow protein (PYP E46Q) are measured using picosecond transient absorption (PTA) spectroscopy. Three intermediates, I 0 E46Q , I 0 qE46Q , and I 1 E46Q , are observed in the room-temperature PYP E46Q photocycle. Although their respective formation and decay rates differ, I 0 E46Q , I 0 qE46Q , and I 1 E46Q correspond to analogous intermediates (i.e., I 0 , I 0 q , and I 1) observed by PTA in the room-temperature photocycle of the wild type (WT) photoactive yellow protein (PYP). These PTA data show that the replacement of glutamic acid 46 with glutamine influences the kinetic properties of the PYP photocycle, but does not alter the general photochemical mechanism itself. The influence of the E46Q mutation on the PYP chromophore can be independently obtained by measuring changes in the vibrational degrees of freedom of ground-state PYP and PYP E46Q. Vibrational spectra (1100-1700 cm-1) of both PYP and PYP E46Q are measured under the same experimental conditions (i.e., ω 1) 490 nm and ω s) 518-535 nm) using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Although the 14 vibrational bands observed in the PR/CARS spectrum of PYP E46Q are generally analogous to those found in the PR/CARS spectrum of PYP, detailed comparisons reveal significant differences in both the positions and relative intensities of vibrational bands assigned to the phenolate part of the cinnamyl chromophore. These PR/CARS results demonstrate that while the chromophore within both PYP and PYP E46Q have similar vibrational degrees of freedom, the E46Q mutation selectively alters the structure of the phenolate ring, apparently through differences in the hydrogen bonding network involving glutamic acid 46 and the negatively charged oxygen in the phenolate ring. When considered together, the changes in the kinetic rate constants for the photocycle (PTA data) and in the vibrational spectra (PR/CARS data) caused by the E46Q mutation suggest that the I 0 and I 0 q intermediates involve structural and/or electronic energy changes localized on the phenolate ring of the PYP chromophore.
The first vibrational spectrum of batho-rhodopsin (Batho), an intermediate formed during the init... more The first vibrational spectrum of batho-rhodopsin (Batho), an intermediate formed during the initial 50 ps of the visual process of rhodopsin (Rh) at room temperature, has been measured by picosecond time-resolved coherent anti-Stokes scattering (PTR/CARS). The Batho spectrum is obtained through a third-order susceptibility (χ(3)) analysis of the measured PTR/CARS data in which the resonant and non-resonant contributions to the signal are separated. The PTR/CARS spectrum of Batho reveals structural changes during the Rh photo-reaction which have not previously been recognized.
Journal of the American Chemical Society, Dec 21, 1999
The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteri... more The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a five-membered carbon ring spanning the C 12-C 13 dC 14 bonds (BR5.12) is measured by picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). Observed initially by picosecond transient absorption (PTA) measurements, T5.12 is the only intermediate in the BR5.12 photoreaction (i.e., T5.12 decays only to BR5.12). BR5.12 does not have a photocycle analogous to that in native BR, presumably because the five-membered ring blocks the reaction coordinate leading to C 13 dC 14 bond isomerization. Since T5.12 may therefore represent the molecular events (reaction coordinates) that precede C 13 dC 14 bond isomerization, its vibrational spectrum may aid in elucidating the primary reaction coordinate-(s) in the BR photocycle. Although T5.12 is identified via a red-shifted absorption (660 nm maximum, <3 ps formation with 3 ps BR5.12 excitation and decay in 17 (1 ps), no spectroscopic data which directly characterize the retinal structure in T5.12, and thereby the role of bonding changes, have been available. The PTR/CARS vibrational data presented here show that T5.12 contains (i) an all-trans retinal configuration, (ii) significant hydrogen out-of-plane motion localized in specific normal modes, (iii) increased π-electron density in the CdC stretching modes manifested by frequency increases, (iv) restricted in-plane C-CH 3 rocking motion, and (v) a Schiff-base environment similar to that in BR5.12. These PTR/CARS data also confirm that T5.12 decays exclusively to BR5.12. The vibrational spectrum of T5.12 makes it evident that no complete CdC isomerization nor CC rotation in any retinal bond occurs upon excitation of BR5.12. The excellent agreement between the kinetic lifetime of T5.12 (from PTA and PTR/CARS data) and its stimulated emission lifetime suggests that T5.12 may be an excited electronic state. In such a case, the PTR/CARS data presented here are the first to be reported from an excited electronic state of a protein. Regardless of whether T5.12 is an excited or ground electronic state, the vibrational spectra of T5.12 reflect the retinal structure(s) that precedes C 13 d C 14 bond isomerization in the BR photocycle. The relevance of T5.12 PTR/CARS data to the native BR photocycle is discussed in terms of the intermediates K-590, J-625, and I-460. Direct analyses of the respective vibrational spectra of T5.12 and K-590 demonstrate that they contain distinctly different retinal structures, but since no vibrational data assignable directly to either I-460 or J-625 have been reported, comparisons of T5.12 with these intermediates are based only on analogy. Comparisons of the vibrational spectra of T5.12 and native BR intermediates independently provide insight into the structural changes in retinal that could occur prior to C 13 dC 14 bond isomerization in native BR.
Bulletin of the American Physical Society, Mar 18, 2013
Florida-It is well known that the generalized Kramers-Kronig relationship is able to retrieve the... more Florida-It is well known that the generalized Kramers-Kronig relationship is able to retrieve the phase of a signal from measured power spectra. This phase recovery is a critical procedure in nonlinear optical spectroscopy, e.g. coherent Raman time domain or frequency domain spectroscopy. Several other methods have been developed and being used in the past: notably, nonlinear fitting and maximum entropy method. A firm mathematical comparison of the methods including the effects of final signal sampling and their merit of fidelity will be presented. Attention is given to numerical implementation of the phase retrieval procedure to put it into practice in coherent anti-Stokes Raman microscopy. Phase retrieval examples using all the above methods are taken from earlier and recently recorded spectra. 1 Financial support from the University of West Florida is acknowledged.
Vibrational spectra from ground-state bacteriorhodopsin (BR) in light-adapted protonated and perd... more Vibrational spectra from ground-state bacteriorhodopsin (BR) in light-adapted protonated and perdeuterated (D20) water samples (i.e. from BR-570) are recorded by picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Coherent excitation by pairs of 6 ps pulses at o, (578 nm, Aw<4 cm-') and at w, (604-643 nm, A~=300 cm-') simultaneously generates PR/CARS signals over = 300 cm-'. The high S/N of the PR/CARS spectra obtained over the 760-1750 cm-' region merit a quantitative xc3' analysis to obtain band origin positions, band shapes, relative band intensities, and phase factors describing the electronic resonance. Excitation conditions are selected to minimize BR photochemistry and ensure the electronic resonance of o, with the retinal chromophore and therefore, these PR/CARS data can be assigned to the all-trans, 15-anti retinal structure found in BR-570. Changes not only in the retinal structure itself, but also in the surrounding environment (protein and water molecules) which either alter the retinal structure or influence the electronic resonance in the retinal chromophore are reflected in these results. Thus, a more detailed view than previously available can be obtained of the vibrational degrees of freedom of all-trans, 15-anti retinal. Relative to resonance Raman data from BR-570, 8 new vibrational features in the PR/ CARS spectra from BR-570 and changes in the positions of Raman bands directly attributable to the deuteration of the Schiff base nitrogen are found. Attention also is given to the influence of non-resonant and resonant contributions to the PR/CARS signal. These results are especially important in the analysis of PR/CARS data from samples in which mixtures of BR retinal chromophores, both static and transient, are found. * Corresponding author. membrane has been shown to involve structural (configurational and conformational) transformations in both the retinal chromophore and in its surrounding protein environment [ 21. The photolytically initiated changes in the electronic absorption maxima assigned to the retinal chromophore have been used to identify two photophysical (H and I) and at least six photochemical (J, K, L, M, N and 0) intermediates which define the BR photocycle [ 31. After several milliseconds, the initial ground-state species, BR-570, is reformed from 0. The fluorescence spectroscopy of
ABSTRACT The molecular mechanism describing the initial 200 ps of the room-temperature photocycle... more ABSTRACT The molecular mechanism describing the initial 200 ps of the room-temperature photocycle in the artificial bacteriorhodopsin (BR) pigment, BR6.9, is examined by both absorption and vibrational spectroscopy. The BR6.9 pigment contains a structurally modified retinal chromophore (retinal 6.9) having a six-membered carbon ring bridging the C9C10−C11 bonds. Picosecond transient absorption (PTA) data show that the initial 200-ps interval of the BR6.9 photocycle contains two intermediates: J6.9 formed with a &lt;3-ps time constant and decaying to K6.9 with a 5-ps time constant (K6.9 has a &gt;5-ns lifetime). Resonantly enhanced vibrational spectra from the light- and dark-adapted ground states of BR6.9 are measured using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Each of these PR/CARS spectra (800−1700 cm-1) contains 33 features assignable to the vibrational degrees of freedom in the retinal chromophore. CARS spectra from the K6.9, obtained from picosecond time-resolved CARS (PTR/CARS) data using 10-ps, 50-ps, 100-ps, and 200-ps time delays following the 570-nm initiation of the BR6.9 photocycle, contain a comparable number of features assignable to the retinal in K6.9. The vibrational spectrum of J6.9 can be tentatively characterized by two bands observed in the 1120−1200-cm-1 region from the analysis of the 10-ps PTR/CARS data. Comparisons involving these PTA and CARS data from BR6.9, as well as analogous results obtained from the ground states and photocycle intermediates of native BR and other artificial BR pigments, demonstrate that restricting retinal motion at the C9C10−C11 bonds does not generally change the initial 200-ps photocycle mechanism, but does alter the rates at which specific molecular processes occur. These vibrational CARS spectra show that the retinal structures in K6.9 and in both light- and dark-adapted BR6.9 are all distinct. However, the specific mechanistic role, if any, of C13C14 isomerization cannot be directly identified from CARS data recorded from BR6.9 and its photocycle intermediates. Even though C13C14 isomerization has been widely considered the primary retinal structural change underlying the proton-pumping mechanism in BR pigments, these results leave open the question of whether C13C14 isomerization is required as a mechanistic precursor for biochemical activity in BR pigments such as BR6.9.
In the final stages of the production process, after the paper had been published, an unfortunate... more In the final stages of the production process, after the paper had been published, an unfortunate error was found in the title: anti-Raman should have been anti-Stokes Raman. Also, the seventh line of the Abstract (633.4 nm) and to s (675-700 nm) should have been (663 nm) and tos (698-745 nm).
Bulletin of the American Physical Society, Jun 1, 2018
Florida-We report the recent progress of construction and characterization of a coherent Raman ta... more Florida-We report the recent progress of construction and characterization of a coherent Raman tabletop system utilizing a novel broadband nanosecond optical parametric oscillator and volumetric Bragg filters assisting a PS-3CBCRS measuring system. To illustrate the versatility of the system and precision of the measurements to reveal the molecular information, we selected two chemicals. Polarization sensitive epi-detected 3CBCRS spectra of liquid CCl 4 and DCVJ were recorded and analyzed. By changing the polarization angles of the actinic laser waves, the vibrational bands resembled the Lorentzian line-shapes extracted from dispersive spectra by non-model dependent spectral processing methods. Electronic resonance enhanced PS-3CBCRS spectra of DCVJ was recorded for the first time. The new outcomes are additions to our recently reported 3CBCRS results on liquid and crystal samples.
Bulletin of the American Physical Society, Mar 23, 2011
The liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was in... more The liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was injected into a commercially available liquid crystal cell (INSTEC, Inc). The cell was housed in a temperature controlled environment constructed in the lab and exposed to light from a frequency doubled Nd: YAG laser. The spectrum of fluorescence from the sample was measured at several temperatures over the range 250 to 450C, covering the smectic-nematic and nematic-isotropic phase transitions. The sample was held at each temperature with a precision and resolution of 1mK before taking the spectrum. It was therefore possible to approach very close to the phase transitions. The results show a significant change in the fluorescence spectrum near the nematic-isotropic phase transition.
Bulletin of the American Physical Society, Mar 3, 2015
Florida-Laser Induced Breakdown Spectroscopy (LIBS) is a very practical spectroscopy to determine... more Florida-Laser Induced Breakdown Spectroscopy (LIBS) is a very practical spectroscopy to determine the chemical composition of materials. Recent technical developments resulted in equipment used on the MARS Rover by NASA. It is capable of measuring the emission spectra of laser induced plasma created by energetic laser pulses focused on the sample (rocks, metals, etc.). We have develop a Laser Induced Breakdown Spectroscopy setup and investigated the necessary experimental and methodological challenges needed to make such material identification measurements. 355 and 532 nm laser pulses with 5 ns temporal duration was used to generate micro-plasma from which compositions can be determined based on known elemental and molecular emission intensities and wavelengths. The performance of LIBS depends on several parameters including laser wavelength, pulse energy, pulse duration, time interval of observation, geometrical configuration of collecting optics, and the properties of ambient medium. Spectra recorded from alloys (e.g. US penny coin) and pure metals will be presented.
Spectral fidelity of any coherent Raman spectra is a major concern of building and/or using an in... more Spectral fidelity of any coherent Raman spectra is a major concern of building and/or using an instrument in "real-time" applications such as e.g. chemical component monitoring in a reaction chamber, or mapping the distribution of certain chemicals in a solid sample matrix. Mainly, three methods are used to recover the vibrational information from the measured spectra: 1. Nonlinear fitting based upon the Levenberg-Marquardt algorithm[1], 2. Maximum Entropy Method[2], and 3. Direct phase retrieval procedure based upon the assumption that the third order susceptibility is an analytic function[3]. Corrections for instrumental response and the problem of finite sampling with appropriate "windowing" are also necessary to recover the vibrational bands needed to identify or characterize the samples. These mathematical methods will be explained and a computer program written in LabView will be presented. Spectra taken by a nanosecond (at UWF)[4] and a picosecond (at UofA)[5] CARS spectrometer will be presented and analyzed by all three methods, vice infra. It is concluded that the third method is the most appropriate one for "real-time" data processing.
relevant to the interpretation of experimental spectra will be presented. The method is applied t... more relevant to the interpretation of experimental spectra will be presented. The method is applied to measure the vibrational manifold of DCVJ for the first time. DCVJ is a charge transfer molecular rotor showing a viscosity dependent fluorescence quantum yield. Based upon the measured CARS spectra, the effect of inhibition of the internal rotation on the vibrational motion of the molecule will be discussed. The design and operation of an all solid-state broadband nanosecond CARS system will be also presented. An overview of applications of molecular rotors in biology and information technology will be outlined.
Bulletin of the American Physical Society, Mar 23, 2011
liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was inject... more liquid crystal 4'octyl-4-cyanobiphenyl (8CB) doped with cadmium selenide nanoparticles was injected into a commercially available liquid crystal cell (INSTEC, Inc). The cell was housed in a temperature controlled environment constructed in the lab and exposed to light from a frequency doubled Nd: YAG laser. Fluorescence from the sample was filtered from the incident light and detected using a photodiode and measured with a lock-in amplifier. Measurements have been made over the temperature range 25 • to 45 • C. The sample was stabilized at each temperature, and the fluorescence intensity was measured at several temperatures. The results show a significant change in fluorescence near the nematic-isotropic phase transition. The temperature control and precision allowed more than 1000 data points to be taken between 25-50 • C, with most of these clustered in the transition region between 38.5-39.5 • C, where the change in intensity was observed.
XXII INTERNATIONAL CONFERENCE ON RAMAN SPECTROSCOPY, 2010
The emerging field of nanotechnology requires new methodologies to be developed in order to addre... more The emerging field of nanotechnology requires new methodologies to be developed in order to address the needs of fabrication, manipulation, control, and measurement of motions of molecules and devices on the nanoscale. Optical excitation and control of translational, vibrational, and rotational motions can offer a unique way to achieve the desired goals by using a sequence of short laser pulses, e.g., tuned to initiate certain nuclear motions of well studied photo active proteins (e.g. PYP or Rhodopsin) or engineered larger molecular structures. Measuring nuclear motions requires structurally sensitive spectroscopy methods, and therefore spontaneous Raman and coherent Raman spectroscopies can be applied to qualitatively test and quantitatively measure such motions. Polarization Sensitive Resonance Coherent anti-Stokes Raman Spectroscopy (PSCARS), steady state or time-resolved, can measure controlled dipole moment reorientation through the anisotropic third order nonlinear optical susceptibility. The measured vibrational spectra are sensitive to photo induced isomerization or internal molecular rotation if the rotating chemical group is coupled to or interacting with internal normal modes or to the surrounding molecules e.g. in solutions. In our study, the method was applied to measure the vibrational manifold and reorientation of DCVJ for the first time. DCVJ is a charge transfer molecular rotor showing a viscosity dependent fluorescence quantum yield. This dependence is attributed to the ``friction'' type interaction between the 90 degree internal rotation of the molecule upon photo-excitation and the solvent cage surrounding it. Quantum chemical studies using DFT and normal mode calculations support our findings related to the conformational evolution of the molecule. Theoretical and experimental principles associated with CARS with an emphasis on points relevant to the interpretation of the results will be presented. The design and operation of an all solid-state broadband nanosecond CARS system will be also presented. Finally, an overview of applications of molecular rotors in biology, nanotechnology and perhaps information technology will be outlined.
Bulletin of the American Physical Society, Mar 15, 2010
capacitance measurement has been performed near the phase transitions of scientifically important... more capacitance measurement has been performed near the phase transitions of scientifically important liquid crystals such as 8-CB. The details of the measurements to get high precision data on dielectric constant and its temperature dependence will be presented. The results show significant changes of the dielectric properties of the liquid crystal near the smectic-to-nematic and nematic-to-liquid phase transitions attributed to structural changes of the relevant phases. In order to measure the details of the functional dependence near the phase transition, the temperature was varied with milliKelvin precision. The data was obtained using a self-assembled RC circuit with phase sensitive lock-in amplifier detection. Calibration of the device was made by measuring known standard capacitances. In order to get high accuracy the measurement was completely computer controlled. The Method applied here will contribute to the better understanding of thermodynamic behavior of liquid crystals and can be routinely used to characterize novel materials showing phase transitions.
Vibrational spectra recorded by coherent antiatokes resonance Raman scattering (CARS) from bacter... more Vibrational spectra recorded by coherent antiatokes resonance Raman scattering (CARS) from bacteriorhodopsin (BR) samples containing isotopically substituted (' H and 13C) retinal chromophores were measured using high repetition rate, low-power, picosecond pulsed excitation (1, = 580 nm and 1, = 640 f 3 nm). These picosecond resonance CARS (PR/CARS) data were analyzed via third-order susceptibility relationships [ ~(~' 1 to obtain band origins, bandwidths, relative intensities, and electronic phase factors assignable to all sipilkant vibrational Raman features in the 1490-1700 cm-' wavenumber region (the ethylenic stretching and C=N-H rocking or Scbiff base modes). Isotopic substitution selectively places ' H at C,, , 13C singly at the C,, position and at the C,, position, and "C simultaneously in positions of C,, and C,,. Each isotopic BR sample was examined not only in H'O, but also in D,O, which places a ' H at the Schiff base nitrogen of the retinal. In addition, PR/CARS data were recorded from each isotopic BR sample following either light adaptation lie. the BR sample contained a single retinal isomer (all-trams, 1 k t i or BR-570)I or dark adaptation 1i.e. the BR sample contained a mixture of comparable amounts of retinal isomers (BR-570 and 13-cis, 15-syn or BR-548)]. Excellent agreement was found between the vibrational features observed by PR/CARS and those obtained from spontaneous resonance Raman measurements from the same isotopically substituted BR pigments. Several new vibrational features were also found from the PR/CARS data. Vibrational Raman data from three of the isotopic BR samples in D,O are reported for the first time.
The dynamics associated with the formation bathorhodopsin (batho) intermediate in the room-temper... more The dynamics associated with the formation bathorhodopsin (batho) intermediate in the room-temperature rhodopsin (RhRT) photosequence are investigated with picosecond transient absorption (PTA) spectroscopy. The transient batho concentration is continuously monitored via absorbance changes with 2-ps time resolution for time delays as long as 500 ps. By resolving both the (5 f 1)-ps formation time constant of batho from photorhodopsin and measuring a constant batho concentration over the 20-500-ps interval, these PTA data bridge earlier studies which measured each of these processes separately but not both simultaneously. The FTA data presented here are compared both with other time-resolved absorption studies from which contradictory kinetic results have been obtained and with recently reported picosecond time-resolved vibrational studies (coherent anti-Stokes Raman) of room-temperature batho.
The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of... more The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of the E46Q mutant of photoactive yellow protein (PYP E46Q) are measured using picosecond transient absorption (PTA) spectroscopy. Three intermediates, I 0 E46Q , I 0 qE46Q , and I 1 E46Q , are observed in the room-temperature PYP E46Q photocycle. Although their respective formation and decay rates differ, I 0 E46Q , I 0 qE46Q , and I 1 E46Q correspond to analogous intermediates (i.e., I 0 , I 0 q , and I 1) observed by PTA in the room-temperature photocycle of the wild type (WT) photoactive yellow protein (PYP). These PTA data show that the replacement of glutamic acid 46 with glutamine influences the kinetic properties of the PYP photocycle, but does not alter the general photochemical mechanism itself. The influence of the E46Q mutation on the PYP chromophore can be independently obtained by measuring changes in the vibrational degrees of freedom of ground-state PYP and PYP E46Q. Vibrational spectra (1100-1700 cm-1) of both PYP and PYP E46Q are measured under the same experimental conditions (i.e., ω 1) 490 nm and ω s) 518-535 nm) using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Although the 14 vibrational bands observed in the PR/CARS spectrum of PYP E46Q are generally analogous to those found in the PR/CARS spectrum of PYP, detailed comparisons reveal significant differences in both the positions and relative intensities of vibrational bands assigned to the phenolate part of the cinnamyl chromophore. These PR/CARS results demonstrate that while the chromophore within both PYP and PYP E46Q have similar vibrational degrees of freedom, the E46Q mutation selectively alters the structure of the phenolate ring, apparently through differences in the hydrogen bonding network involving glutamic acid 46 and the negatively charged oxygen in the phenolate ring. When considered together, the changes in the kinetic rate constants for the photocycle (PTA data) and in the vibrational spectra (PR/CARS data) caused by the E46Q mutation suggest that the I 0 and I 0 q intermediates involve structural and/or electronic energy changes localized on the phenolate ring of the PYP chromophore.
The first vibrational spectrum of batho-rhodopsin (Batho), an intermediate formed during the init... more The first vibrational spectrum of batho-rhodopsin (Batho), an intermediate formed during the initial 50 ps of the visual process of rhodopsin (Rh) at room temperature, has been measured by picosecond time-resolved coherent anti-Stokes scattering (PTR/CARS). The Batho spectrum is obtained through a third-order susceptibility (χ(3)) analysis of the measured PTR/CARS data in which the resonant and non-resonant contributions to the signal are separated. The PTR/CARS spectrum of Batho reveals structural changes during the Rh photo-reaction which have not previously been recognized.
Journal of the American Chemical Society, Dec 21, 1999
The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteri... more The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a five-membered carbon ring spanning the C 12-C 13 dC 14 bonds (BR5.12) is measured by picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). Observed initially by picosecond transient absorption (PTA) measurements, T5.12 is the only intermediate in the BR5.12 photoreaction (i.e., T5.12 decays only to BR5.12). BR5.12 does not have a photocycle analogous to that in native BR, presumably because the five-membered ring blocks the reaction coordinate leading to C 13 dC 14 bond isomerization. Since T5.12 may therefore represent the molecular events (reaction coordinates) that precede C 13 dC 14 bond isomerization, its vibrational spectrum may aid in elucidating the primary reaction coordinate-(s) in the BR photocycle. Although T5.12 is identified via a red-shifted absorption (660 nm maximum, <3 ps formation with 3 ps BR5.12 excitation and decay in 17 (1 ps), no spectroscopic data which directly characterize the retinal structure in T5.12, and thereby the role of bonding changes, have been available. The PTR/CARS vibrational data presented here show that T5.12 contains (i) an all-trans retinal configuration, (ii) significant hydrogen out-of-plane motion localized in specific normal modes, (iii) increased π-electron density in the CdC stretching modes manifested by frequency increases, (iv) restricted in-plane C-CH 3 rocking motion, and (v) a Schiff-base environment similar to that in BR5.12. These PTR/CARS data also confirm that T5.12 decays exclusively to BR5.12. The vibrational spectrum of T5.12 makes it evident that no complete CdC isomerization nor CC rotation in any retinal bond occurs upon excitation of BR5.12. The excellent agreement between the kinetic lifetime of T5.12 (from PTA and PTR/CARS data) and its stimulated emission lifetime suggests that T5.12 may be an excited electronic state. In such a case, the PTR/CARS data presented here are the first to be reported from an excited electronic state of a protein. Regardless of whether T5.12 is an excited or ground electronic state, the vibrational spectra of T5.12 reflect the retinal structure(s) that precedes C 13 d C 14 bond isomerization in the BR photocycle. The relevance of T5.12 PTR/CARS data to the native BR photocycle is discussed in terms of the intermediates K-590, J-625, and I-460. Direct analyses of the respective vibrational spectra of T5.12 and K-590 demonstrate that they contain distinctly different retinal structures, but since no vibrational data assignable directly to either I-460 or J-625 have been reported, comparisons of T5.12 with these intermediates are based only on analogy. Comparisons of the vibrational spectra of T5.12 and native BR intermediates independently provide insight into the structural changes in retinal that could occur prior to C 13 dC 14 bond isomerization in native BR.
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