This work was supported by JSPS KAKENHI Grant Numbers JP24750023, JP24350012, MEXT KAKENHI Grant Numbers JP26104534, JP16H00850, JP26102541, JP16H00782, and MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015&#8211;2019.

<ol>
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The authors have nothing to disclose.

Crucial factors in femtosecond time-resolved near-IR multiplex stimulated Raman measurement 
To obtain time-resolved near-IR stimulated Raman spectra with a high signal-to-noise ratio, the probe spectrum should ideally have uniform intensity in the whole wavelength range. White-light continuum generation (section 2.5) is, therefore, one of the most crucial parts of time-resolved near-IR stimulated Raman experiments. In general, the probe spectrum becomes broad and flat as the intensity of the incident beam increases. A high beam intensity, however, easily produces undesirable nonlinear optical effects other than the white light continuum generation. In a worst-case scenario, the nonlinear effects provide the probe spectrum with a large intensity fluctuation and an oscillatory pattern that significantly lowers the signal-to-noise ratio of stimulated Raman spectra. Figure 2C shows how the oscillatory pattern affects the spectra. It shows oscillatory patterns from -0.30 to 4 ps, but the patterns appear only weakly, with a peak-to-peak amplitude of 1 x 10-4, as white light generation is carefully optimized. Another undesirable effect on the probe spectrum can be provided by water vapor in the air2,11. The effect of water vapor might be avoided if part of the spectrometer, including the white light generation optics, sample, and spectrograph, is set in a chamber filled with dry nitrogen.
Accuracy of Raman shift calibration 
As described in section 8, we calibrate the Raman shift axis by the least-squares fitting analysis of the peak positions of the solvent bands in Raman shift against those in the pixel number of the detector with a polynomial function. We think this protocol works well as long as the Raman pump wavelength cannot be determined with high accuracy. It is the case for our spectrometer because each pixel of our detector covers as large as 3.5 cm-1 at around the wavenumber of the Raman pump pulse. However, the solvents must be chosen so that all the transient stimulated Raman bands of the sample appear between the highest and lowest wavenumbers of the solvent bands (section 8). The Raman shift calibration curve loses its accuracy beyond the range of the solvent bands. In Figure 2, a Raman band of S1 &#946;-carotene in toluene, at 1,785 cm-1, appears beyond the highest wavenumber of the solvent bands, 1,710 cm-1. We have confirmed that the peak position agrees well with that in benzene determined by picosecond time-resolved spontaneous Raman spectroscopy35,36.
Effectiveness and perspective of femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer 
It has been demonstrated that the femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer can observe stimulated Raman spectra, which provides information almost equivalent to spontaneous Raman spectra of short-lived species with near-IR transitions. Small differences in the peak position of a band can be detected with the spectrometer because of its sufficiently high sensitivity. The spectrometer will be applicable to a wide variety of &#960;-conjugate systems from simple aromatic molecules to photoconductive polymers. Stationary near-IR multiplex stimulated Raman spectroscopy is also a powerful tool for observing molecular vibrations without fluorescence interference from the sample, because the energy of near-IR photons is generally much lower than the electronic transition energy of molecules from the lowest excited singlet state to the ground state. The spectrometer will be applicable to in vivo observation of the structural dynamics in biological systems.

Raman spectroscopy is a powerful and versatile tool for investigating the structures of molecules in a wide variety of samples from simple gases, liquids, and solids to functional materials and biological systems. Raman scattering is significantly enhanced when the photon energy of the excitation light coincides with the electronic transition energy of a molecule. The resonance Raman effect enables us to selectively observe the Raman spectrum of a species in a sample composed of many kinds of molecules. Near-IR electronic transitions are drawing a lot of attention as a probe for investigating the excited-state dynamics of molecules with large &#960;-conjugated structures. The energy and lifetime of the lowest excited singlet state have been determined for several carotenoids, which have a long one-dimensional polyene chain1,2,3. The dynamics of neutral and charged excitations have been extensively investigated for various photoconductive polymers in films4,5,6,7, nanoparticles8, and solutions9,10,11. Detailed information on the structures of the transients will be obtainable if time-resolved near-IR Raman spectroscopy is applied to these systems. Only a few studies, however, have been reported on time-resolved near-IR Raman spectroscopy12,13,14,15,16, because the sensitivity of near-IR Raman spectrometers is extremely low. The low sensitivity principally originates from the low probability of near-IR Raman scattering. The probability of spontaneous Raman scattering is proportional to &#969;i&#969;s3, where &#969;i and &#969;s are the frequencies of the excitation light and the Raman scattering light, respectively. In addition, commercially available near-IR detectors have much lower sensitivity than CCD detectors functioning in the UV and visible regions.
Femtosecond time-resolved stimulated Raman spectroscopy has emerged as a new method of observing time-dependent changes of Raman active vibrational bands beyond the apparent Fourier-transform limit of a laser pulse17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33. Stimulated Raman scattering is generated by irradiation of two laser pulses: the Raman pump and probe pulses. Here it is assumed that the Raman pump pulse has a larger frequency than the probe pulse. When the difference between the frequencies of the Raman pump and probe pulses coincides with the frequency of a Raman active molecular vibration, the vibration is coherently excited for a large number of molecules in the irradiated volume. Nonlinear polarization induced by the coherent molecular vibration enhances the electric field of the probe pulse. This technique is particularly powerful for near-IR Raman spectroscopy, because stimulated Raman scattering can solve the problem of the sensitivity of time-resolved near-IR spontaneous Raman spectrometers. Stimulated Raman scattering is detected as intensity changes of the probe pulse. Even if a near-IR detector has a low sensitivity, stimulated Raman scattering will be detected when the probe intensity is sufficiently increased. The probability of stimulated Raman scattering is proportional to &#969;RP&#969;SRS, where &#969;RP and &#969;SRS are the frequencies of the Raman pump pulse and stimulated Raman scattering, respectively20. The frequencies for stimulated Raman scattering, &#969;RP and &#969;SRS, are equivalent to &#969;i and &#969;s for spontaneous Raman scattering, respectively. We have recently developed a femtosecond time-resolved near-IR Raman spectrometer using stimulated Raman scattering for investigating the structures and dynamics of short-lived transients photogenerated in &#960;-conjugate systems2,3,7,10. In this article, we present the technical details of our femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer. Optical alignment, acquisition of time-resolved stimulated Raman spectra, and calibration and correction of recorded spectra are described. The excited-state dynamics of &#946;-carotene in toluene solution is studied as a representative application of the spectrometer.

<table><tbody><tr><td>1-Axis Translational Stage</td><td>OptSigma</td><td>TSD-401S</td><td>Products equivalent to this are used as well; for M22, L9, and CM in Figure 1A</td></tr><tr><td>20-cm Optical Delay Line</td><td>OptSigma</td><td>SGSP26-200</td><td>ODL1 in Figure 1A</td></tr><tr><td>3-Axis Translational Stage</td><td>OptSigma</td><td>TSD-405SL</td><td>For L8 in Figure 1A</td></tr><tr><td>3-Axis Translational Stage</td><td>Suruga Seiki</td><td>B72-40C</td><td>For FC in Figure 1A</td></tr><tr><td>5-cm Optical Delay Line</td><td>PMT</td><td>HRS-0050</td><td>ODL2 in Figure 1A</td></tr><tr><td>Al Concave Mirror</td><td>Thorlabs</td><td>CM254-050-G01</td><td>Focal length: 50 mm; CM in Figure 1A</td></tr><tr><td>Base Plate</td><td>Suruga Seiki</td><td>A21-6</td><td>Products equivalent to this are used as well; for M1-M32, BS1-BS3, L1-L10, I1-I17, P1-P2, HWP1-3, F1-F3, VND1-VND2, OC, BPF, HS, BBO, SP, CM, and FC in Figure 1A</td></tr><tr><td>BBO Crystal</td><td>EKSMA Optics</td><td>-</td><td>Type 1, &amp;theta; = 23.2 deg; BBO in Figure 1A</td></tr><tr><td>BK7 Plano-Concave Lens</td><td>OptSigma</td><td>SLB-25.4-50NIR2</td><td>Focal length: 50 mm; IR anti-reflection coating; L6 in Figure 1A</td></tr><tr><td>BK7 Plano-Convex Lens</td><td>OptSigma</td><td>SLB-25.4-150PIR2</td><td>Focal length: 150 mm; IR anti-reflection coating; L2, L3, L5 in Figure 1A</td></tr><tr><td>BK7 Plano-Convex Lens</td><td>OptSigma</td><td>SLB-25.4-100PIR2</td><td>Focal length: 100 mm; IR anti-reflection coating; L4 in Figure 1A</td></tr><tr><td>BK7 Plano-Convex Lens</td><td>OptSigma</td><td>SLB-25.4-200PIR2</td><td>Focal length: 200 mm; IR anti-reflection coating; L7 in Figure 1A</td></tr><tr><td>Broadband Dielectric Mirror</td><td>OptSigma</td><td>TFMS-25.4C05-2/7</td><td>M22-M25, M28, M29 in Figure 1A</td></tr><tr><td>Broadband Dielectric Mirror</td><td>Precision Photonics (Advanced Thin Films)</td><td>-</td><td>M26, M27, M30-M32 in Figure 1A</td></tr><tr><td>Broadband Half-Wave Plate</td><td>CryLight</td><td>-</td><td>HWP3 in Figure 1A</td></tr><tr><td>Color Glass Filter</td><td>HOYA</td><td>IR85</td><td>F1 in Figure 1A</td></tr><tr><td>Color Glass Filter</td><td>HOYA</td><td>RM100</td><td>F2 in Figure 1A</td></tr><tr><td>Color Glass Filter</td><td>Schott</td><td>BG39</td><td>F3 in Figure 1A</td></tr><tr><td>Computer</td><td>Dell</td><td>Vostro 200 Mini Tower</td><td>OS: Windows XP</td></tr><tr><td>Cyclohexane</td><td>Kanto Kagaku</td><td>07547-1B</td><td>HPLC grade</td></tr><tr><td>Data Analysis Software</td><td>Wavemetrics</td><td>Igor Pro 8</td><td></td></tr><tr><td>Dielectric Beamsplitter</td><td>LAYERTEC</td><td>-</td><td>Reflection : Transmission = 2 : 1; BS1 in Figure 1A</td></tr><tr><td>Dielectric Beamsplitter</td><td>LAYERTEC</td><td>-</td><td>Reflection : Transmission = 1 : 1; BS2, BS3 in Figure 1A</td></tr><tr><td>Dielectric Mirror</td><td>Precision Photonics&lt;br/&gt; (Advanced Thin Films)</td><td>-</td><td>M1-M8 in Figure 1A</td></tr><tr><td>Digital Oscilloscope</td><td>Tektronix</td><td>TDS3054B</td><td>500 MHz, 5 GS/s</td></tr><tr><td>Elastomer Tube</td><td>-</td><td>-</td><td>Figure 1E</td></tr><tr><td>Femtosecond Ti:sapphire Oscillator</td><td>Coherent</td><td>Vitesse 800-2</td><td>Wavelength: 800 nm, pulse duration: 100 fs, average power: 280 mW, repetition rate: 80 MHz; included in Ti:S in Figure 1A</td></tr><tr><td>Femtosecond Ti:sapphire Regenerative Amplifier</td><td>Coherent</td><td>Legend-Elite-F-HE</td><td>Wavelength: 800 nm, pulse duration: 100 fs, pulse energy: 3.5 mJ, repetition rate: 1 kHz; included in Ti:S in Figure 1A</td></tr><tr><td>Film Polarizer</td><td>OptSigma</td><td>SPFN-30C-26</td><td>P1 in Figure 1A</td></tr><tr><td>Glan-Taylor Prism</td><td>OptSigma</td><td>GYPB-10-10SN-3/7</td><td>P2 in Figure 1A</td></tr><tr><td>Gold Mirror</td><td>OptSigma</td><td>TFG-25C05-10</td><td>M9-M21 in Figure 1A</td></tr><tr><td>Half-Wave Plate</td><td>OptSigma</td><td>WPQ-7800-2M</td><td>HWP1 in Figure 1A</td></tr><tr><td>Harmonic Separator</td><td>Coherent</td><td>TOPAS-C HRs 410-540 nm</td><td>HS in Figure 1A</td></tr><tr><td>InGaAs Array Detector</td><td>Horiba</td><td>Symphony-IGA-512X1-50-1700-1LS</td><td>512 ch, Liquid nitrogen cooled</td></tr><tr><td>InGaAs PIN Photodiode</td><td>Hamamatsu Photonics</td><td>G10899-01K</td><td></td></tr><tr><td>IR Half-Wave Plate</td><td>OptiSource</td><td>-</td><td>HWP2 in Figure 1A</td></tr><tr><td>Iris</td><td>Suruga Seiki</td><td>F74-3N</td><td>Products equivalent to this are used as well; I1-I17 in Figure 1A</td></tr><tr><td>Lens Holder</td><td>OptSigma</td><td>LHF-25.4S</td><td>Products equivalent to this are used as well; for L1-L10 in Figure 1A</td></tr><tr><td>Magnetic Gear Pump</td><td>Micropump</td><td>184-415</td><td></td></tr><tr><td>Mirror Mount</td><td>Siskiyou</td><td>IM100.C2M6R</td><td>Products equivalent to this are used as well; for M1-M32, BS1-BS3, BBO, CM in Figure 1A</td></tr><tr><td>near-IR phosphor card</td><td>Thorlabs</td><td>VRC2</td><td></td></tr><tr><td>Nut</td><td>-</td><td>-</td><td>Figure 1E, M4; purchased from a DIY store</td></tr><tr><td>Optical Chopper</td><td>New Focus</td><td>3501</td><td>OC in Figure 1A</td></tr><tr><td>Optical Parametric Amplifier</td><td>Coherent</td><td>OPerA-F</td><td>OPA1 in Figure 1A</td></tr><tr><td>Optical Parametric Amplifier</td><td>Coherent</td><td>TOPAS-C</td><td>OPA2 in Figure 1A</td></tr><tr><td>Polarizer Holder</td><td>OptSigma</td><td>PH-30-ARS</td><td>Products equivalent to this are used as well; for P1-P2 and HWP1-3 In Figure 1A</td></tr><tr><td>Polyfluoroacetate Tube</td><td>-</td><td>-</td><td>Figure 1E</td></tr><tr><td>Post Holder</td><td>OptSigma</td><td>BRS-12-80</td><td>Products equivalent to this are used as well; for M1-M32, BS1-BS3, L1-L10, I1-I17, P1-P2, HWP1-3, F1-F3, VND1-VND2, OC, BPF, HS, BBO, SP, CM, and FC in Figure 1A</td></tr><tr><td>Quartz Flow Cell</td><td>Tosoh Quartz</td><td>T-70-UV-2</td><td>FC in Figure 1A</td></tr><tr><td>Quartz Plano-Concave Lens</td><td>OptSigma</td><td>SLSQ-25-50N</td><td>Focal length: 50 mm; L8 in Figure 1A</td></tr><tr><td>Quartz Plano-Convex Lens</td><td>OptSigma</td><td>SLSQ-25-100P</td><td>Focal length: 100 mm; L1, L9 in Figure 1A</td></tr><tr><td>Quartz Plano-Convex Lens</td><td>OptSigma</td><td>SLSQ-25-220P</td><td>Focal length: 220 mm; L10 in Figure 1A</td></tr><tr><td>Sapphire Plate</td><td>Pier Optics</td><td>-</td><td>3 mm thick; SP in Figure 1A</td></tr><tr><td>Si PIN Photodiode</td><td>Hamamatsu Photonics</td><td>S3883</td><td></td></tr><tr><td>Single Spectrograph</td><td>Horiba Jobin Yvon</td><td>iHR320</td><td>Focal length: 32 cm</td></tr><tr><td>Stainless Steel Rod</td><td>Suruga Seiki</td><td>A41-100</td><td>Products equivalent to this are used as well; for M1-M32, BS1-BS3, L1-L10, I1-I17, P1-P2, HWP1-3, F1-F3, VND1-VND2, OC, BPF, HS, BBO, SP, CM, and FC in Figure 1A</td></tr><tr><td>Stainless Steel Rod</td><td>Newport</td><td>J-SP-2</td><td>Figure 1E</td></tr><tr><td>Toluene</td><td>Kanto Kagaku</td><td>40180-1B</td><td>HPLC grade</td></tr><tr><td>U-Shaped Steel Plate</td><td>-</td><td>-</td><td>Figure 1E; purchased from a DIY store</td></tr><tr><td>Variable Neutral Density Filter (with a holder)</td><td>OptSigma</td><td>NDHN-100</td><td>VND1 in Figure 1A</td></tr><tr><td>Variable Neutral Density Filter (with a holder)</td><td>OptSigma</td><td>NDHN-U100</td><td>VND2 in Figure 1A</td></tr><tr><td>Visual Programming Language</td><td>National Instruments</td><td>LabVIEW 2009</td><td>The control software in this study is programmed in LabVIEW 2009</td></tr><tr><td>Volume-Grating Bandpass Filter</td><td>OptiGrate</td><td>BPF-1190</td><td>BPF in Figure 1A</td></tr><tr><td>&amp;beta;-Carotene</td><td>Wako Pure Chemical Industries</td><td>035-05531</td><td></td></tr></tbody></table>

ultrafast time-resolved near-ir stimulated raman measurements of functional &#960;-conjugate systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Femtosecond time-resolved near-IR stimulated Raman spectroscopy was applied to &#946;-carotene in toluene solution. The concentration of the sample was 1 x 10-4 mol dm-3. The sample was photoexcited by the actinic pump pulse at 480 nm with a pulse energy of 1 &#956;J. Time-resolved stimulated Raman spectra of &#946;-carotene in toluene are shown in Figure 2A. The raw spectra contained strong Raman bands of the solvent toluene and a weak Raman band of &#946;-carotene in the ground state as well as Raman bands of photoexcited &#946;-carotene. They were subtracted using the stimulated Raman spectrum of the same solution at 1 ps before photoexcitation. The spectra after the subtraction (Figure 2B) showed distorted baselines that are caused by absorption of photoexcited &#946;-carotene and/or other nonlinear optical processes. The baselines became flat after they were corrected with polynomial functions (Figure 2C).
The time-resolved stimulated Raman spectra of &#946;-carotene showed two strong bands in the 1,400&#8211;1,800 cm-1 region (Figure 2C). A broad stimulated Raman band at 0 ps was assigned to the in-phase C=C stretch vibration of S2 &#946;-carotene. Its peak position was estimated to be 1,556 cm-1. The in-phase C=C stretch band of S1 &#946;-carotene appeared as the S2 C=C stretch band decayed. The peak position of the S1 C=C stretch band was upshifted by 8 cm-1 from 0.12 to 5 ps (Figure 2D). The time constant of the upshift was estimated to be 0.9 ps. The upshift originates from vibrational energy redistribution in S1 &#946;-carotene2,3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60437/60437fig1a.jpg" /> 
Figure 1: Instrument diagrams. (A) The block diagram of a femtosecond time-resolved near-IR stimulated Raman spectrometer. Ti:S = Mode-locked Ti:sapphire laser system; BS = Beamsplitter; OPA = Optical parametric amplifier; BBO = &#946;-Barium borate crystal; OC = Optical chopper; ODL = Optical delay line; BPF = Volume-grating reflective bandpass filter; SP = Sapphire plate; FC = Flow cell; M = Mirror; CM = Concave mirror; L = Lens; I = Iris; P = Polarizer; HWP = Half-wave plate; F = Color glass filter; VND = Variable optical density filter. The figure is adapted from Takaya11 with permission from the PCCP Owner Societies. (B) Four configurations of a mirror mount. V, H, and S represent the vertical adjustment knob, horizontal adjustment knob, and support, respectively. See section 2.1 for details. (C) A schematic diagram of laser beam alignment. m = Mirror; i = Iris. See section 2.3 for details. (D) A schematic diagram of optical delay line alignment. m = Mirror; i = Iris. See section 2.4 for details. (E) Structure of a flow cell mount. See section 2.9 for details. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60437/60437fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60437/60437fig2a.jpg" /> 
Figure 2: Femtosecond time-resolved near-IR stimulated Raman spectra. (A) Femtosecond time-resolved near-IR stimulated Raman spectra of &#946;-carotene in toluene with the actinic pump wavelength at 480 nm. Raman bands of toluene and &#946;-carotene in the ground state are denoted with circles and a triangle, respectively. (B) Femtosecond time-resolved near-IR stimulated Raman spectra of &#946;-carotene in toluene after the Raman bands of toluene and &#946;-carotene in the ground state are subtracted. The baselines of the spectra were fitted with polynomial functions (broken traces). (C) Femtosecond time-resolved near-IR stimulated Raman spectra of &#946;-carotene in toluene after the baseline correction. (D) The peak positions of the in-phase C=C stretch band in the S1 state plotted against the time delay. The C=C stretch bands were fitted with a Gaussian function for estimating their peak positions. The best fitted curve for the shift of the S1 C=C stretch band (solid trace) was obtained by the least-squares fitting analysis with an exponential function. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60437/60437fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional Ï€-conjugate Systems at JoVE.com

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

ultrafast-time-resolved-near-ir-stimulated-raman-measurements

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.1K Views.  Gakushuin University. Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of β-carotene in toluene is shown as a representative application.

Video: Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional π-conjugate Systems

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

Bioengineering

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

Engineering

Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the presence of carbon nanotubes. These nanowires are extreme in the sense that they are the limit of miniaturization of nanowires and their behavior is not always a simple extrapolation of the behavior of larger nanowires as their diameter decreases. The paper then describes the methods required to obtain Raman spectra from extreme nanowires and the fact that due to the van Hove singularities that 1D systems exhibit in their optical density of states, that determining the correct choice of photon excitation energy is critical. It describes the techniques required to determine the photon energy dependence of the resonances observed in Raman spectroscopy of 1D systems and in particular how to obtain measurements of Raman cross-sections with better than 8% noise and measure the variation in the resonance as a function of sample temperature. The paper describes the importance of ensuring that the Raman scattering is linearly proportional to the intensity of the laser excitation intensity. It also describes how to use the polarization dependence of the Raman scattering to separate Raman scattering of the encapsulated 1D systems from those of other extraneous components in any sample.

The paper describes a method for producing extreme nanowires by melt infiltration into carbon nanotubes and how 1D systems may be characterized and investigated using Resonance Raman Spectroscopy to determine vibrational and optical excitation energies.

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Measurement of Ultrafast Vibrational Coherences in Polyatomic Radical Cations with Strong-Field Adiabatic Ionization

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the wavelength of the strong-field ionizing pump pulse from the commonly used 800 nm into the near-infrared (1200-1600 nm), the contribution of adiabatic electron tunneling to the ionization process increases relative to multiphoton absorption. Adiabatic ionization results in predominant population of the ground electronic state of the ion upon electron removal, which effectively prepares a coherent vibrational state (&#34;wave packet&#34;) amenable to subsequent excitation. In our experiments, the coherent vibrational dynamics are probed with a weak-field 800 nm pulse and the time-dependent yields of dissociation products measured in a time-of-flight mass spectrometer. We present the measurements on the molecule dimethyl methylphosphonate (DMMP) to illustrate how using 1500 nm pulses for excitation enhances the amplitude of coherent oscillations in ion yields by a factor of 10 as compared to 800 nm pulses. This protocol may be implemented in existing pump-probe setups through the incorporation of an optical parametric amplifier (OPA) for wavelength conversion.

We present a protocol for probing ultrafast vibrational coherences in polyatomic radical cations that result in molecular dissociation.

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural tunability. However, the progression of MOF-based photochemistry has been hindered by the difficulty in spectrally characterizing these materials. Given that MOFs are typically larger than 100 nm in size, they are prone to excessive light scatter, thereby rendering data from valuable analytical tools like transient absorption and emission spectroscopy nearly uninterpretable. To gain meaningful insights of MOF-based photo-chemical and physical processes, special consideration must be taken toward properly preparing MOFs for spectroscopic measurements, as well as the experimental setups that garner higher quality data. With these considerations in mind, the present guide provides a general approach and set of guidelines for the spectroscopic investigation of MOFs. The guide addresses the following key topics: (1) sample preparation methods, (2) spectroscopic techniques/measurements with MOFs, (3) experimental setups, (3) control experiments, and (4) post-run stability characterization. With appropriate sample preparation and experimental approaches, pioneering advancements toward the fundamental understanding of light-MOF interactions are significantly more attainable.

Here, we use a polymer stabilizer to prepare metal-organic framework (MOF) suspensions that exhibit markedly decreased scatter in their ground-state and transient absorption spectra. With these MOF suspensions, the protocol provides various guidelines to characterize the MOFs spectroscopically to yield interpretable data.

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural ...

Exploring Multiphase Polymeric Systems by Nanoscale Infrared Spectroscopy

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition is commonly assessed using infrared spectroscopy, which provides an average fingerprint of the various materials contained in the volume probed. However, this approach does not offer any details on the arrangement of the phases in the material. Interfacial regions between two polymeric phases, often in the nanoscale range, are also challenging to access. Photothermal nanoscale infrared spectroscopy monitors the local response of materials excited by infrared light with the sensitive probe of an atomic force microscope (AFM). While the technique is suitable for interrogating small features, such as individual proteins on pristine gold surfaces, the characterization of three-dimensional multicomponent materials is more elusive. This is due to a relatively large volume of material undergoing photothermal expansion, defined by the laser focalization onto the sample and by the thermal properties of the polymeric constituents, compared to the nanoscale region probed by the AFM tip. Using a polystyrene (PS) bead and a polyvinyl alcohol (PVA) film, we evaluate the spatial footprint of photothermal nanoscale infrared spectroscopy for surface analysis as a function of the position of PS in the PVA film. The effect of the feature position on the nanoscale infrared images is investigated, and spectra are acquired. Some perspectives on the future advances in the field of photothermal nanoscale infrared spectroscopy are provided, considering the characterization of complex systems with embedded polymeric structures.

Author Spotlight: Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples.

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition ...

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional π-conjugate Systems

Summary

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Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems