Name: direct comparison of hyperspectral stimulated raman scattering and coherent anti-stokes raman scattering microscopy for chemical imaging
Uploaded: 2022-04-28T13:20:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging at JoVE.com

This research was supported by the Purdue University Department of Chemistry startup fund.

1. Instrumental setup for hyperspectral CRS imaging
NOTE: The generation of CRS signal requires the use of high-power (i.e., class 3B or class 4) lasers. Safety protocols must be addressed and proper personal protective equipment (PPE) must be worn at all times when working at such high peak powers. Consult proper documentation before experimentation. This protocol focuses on designing the beam path, chirping the femtosecond pulses, and optimizing imaging conditions. A general optical layout of this hyperspectral CRS microscope is shown in Figure 1. The configuration shown here is one of many existing configurations for CRS microscopy. The CRS microscopy system used in this protocol is built upon a dual-output femtosecond laser source and a laser scanning microscope.
<ol>
	<li>Ensure that the laser source provides two femtosecond pulse trains (120 fs width) with a repetition rate of 80 MHz, including a fixed wavelength at 1,045 nm used as the Stokes beam, and a tunable wavelength from 680 to 1,300 nm used as the pump beam. Synchronize the output pulses with an optical delay difference. Use a microscope frame to construct the imaging platform.</li>
	<li>Designing the beam path
	<ol>
		<li>To control the laser power at the sample, use a half-wave plate and polarization beam splitter (PBS) combination for each laser beam.</li>
		<li>Install an acousto-optic modulator (AOM) in the Stokes laser beam path. Focus the beam with a 150 mm focal length lens into the AOM and recollimate the 0th order output with a 400 mm focal length lens.</li>
		<li>Use the same lens pair (150 mm and 400 mm focal lengths) to expand the pump beam to match the laser beam size with the Stokes.</li>
		<li>Place the 400 mm focal length lenses in both the pump and Stokes beam paths on separate one-dimensional translation stages for fine-tuning the beam divergence and optimizing the beam size before entering the microscope.</li>
		<li>Direct the pump beam with a right-angle reflecting mirror mounted on a motorized translation stage for optical delay tuning. If the Stokes beam needs optical delay, place these components instead into its beam path.</li>
		<li>Allow both beams to be combined at a dichroic mirror with a cutoff wavelength at ~1,000 nm (between the pump and Stokes wavelengths), so that the Stokes beam will transmit through the dichroic mirror while the pump beam is reflected by the dichroic mirror. Send the collinearly combined laser beams to the microscope.</li>
		<li>To chirp the pump and Stokes beams, place glass rods in their beam paths. See step 1.5 for details.</li>
		<li>To confirm proper alignment and beam size, use iris diaphragms after the dichroic mirror and before the microscope. Specifically, install one at a position close to and the other at a distance from the dichroic mirror to confirm good alignment and beam overlap. Use an IR-viewing card or IR viewer to visualize the beam during alignment.</li>
		<li>Use a fast PD and an oscilloscope to approximately measure the optical delay between the pump and Stokes pulses. Trigger the oscilloscope by sampling the laser pulse train.</li>
		<li>Block the pump beam and sample the Stokes beam. Zoom in on one of the pulses and place a vertical cursor on it to mark its temporal location on the oscilloscope.</li>
		<li>Unblock the pump beam and block the Stokes beam. Translate the delay stage until the sample pump pulses temporally align with the marked position.</li>
	</ol>
	</li>
	<li>The laser scanning microscope
	<ol>
		<li>For an upright microscope configuration, send the combined laser beams through a periscope to climb to an appropriate level before reaching the 2D galvo scanning mirrors.</li>
		<li>Measure the laser beam size before the microscope, and set up the proper lens pair after the galvo mirrors to expand the laser beam to best match the size of the entrance pupil of the objective lens.</li>
		<li>Construct a 4-f system using the two lenses, with the back aperture of the objective lens and the center of the two galvo mirrors being conjugate planes. Alternatively, use two separate 1D galvo mirrors with two 4-f lens systems for laser scanning.</li>
		<li>After the condenser, design a 2 in flip mirror to reflect the laser beams for signal collection. Position a lens with a 2 in diameter in the transmitted beam path to fully collect and focus transmission signals to the detectors.</li>
		<li>Direct the CARS signals to the PMT with a dichroic mirror having a cutoff at 776 nm, and allow the transmitted SRS signals to be detected by the PD. Use a bandpass filter (655/30 nm) before the PMT to reject residual excitation laser pulses. Use a short-pass filter (980 nm short pass) before the PD to block the Stokes beam from entering the detector.</li>
		<li>For CARS signal detection, connect a preamplifier and a current-voltage converter after the PMT and before sending the signal to the data acquisition system. Adjust the PMT voltage to optimize the signal and image contrast.</li>
		<li>Use a function generator to modulate the AOM at 1-10 MHz, and use the same frequency as the reference for the lock-in demodulation. Use a lock-in amplifier to extract SRS signals before data acquisition.</li>
	</ol>
	</li>
	<li>Data acquisition and display
	<ol>
		<li>Perform data acquisition using a digital data acquisition (DAQ) card in conjunction with a terminal block.</li>
		<li>Use the analog outputs from the DAQ to control the galvo mirrors and the analog inputs for signal acquisition.</li>
		<li>Use Lab-written software based on LabVIEW having a simultaneous multichannel display for real-time viewing and saving images (see Supplemental File).</li>
	</ol>
	</li>
	<li>Chirping the femtosecond source and measuring the spectral resolution 
	NOTE: To achieve a good spectral resolution using spectral focusing, glass rods are used to introduce dispersions and chirp laser pulses from femtosecond to picosecond. To achieve the best spectral resolution, the chirp rate of the pump beam needs to equal that of the Stokes beam. For this laser system, the best spectral resolution can be achieved by chirping the ~120 fs output laser pulses to 3.4 ps for the pump and 1.8 ps for the Stokes. This chirping is achieved by using a 4+1 combination (four in the combined beam, one only in the Stokes beam) of 150 mm glass rod (SF-57) combination, as described below, and should achieve a 15 cm-1 spectral resolution. The pulse duration can be measured using an autocorrelator.
	<ol>
		<li>Insert one 150 mm glass rod into only the Stokes beam path.</li>
		<li>Insert two 150 mm glass rods into the combined pump/Stokes beam path after the dichroic beam splitter. To increase the chirping, let the combined laser beams double-pass the two glass rods by placing a dielectric mirror at one end of the rods.</li>
		<li>To measure the spectral resolution, prepare a standard chemical (e.g., dimethyl sulfoxide [DMSO]) sample pressed between two glass coverslips, and scan the delay stage until the maximum signal is achieved.</li>
		<li>Move the optical delay 1,000 &#181;m in the red-shift direction. Then, run 200 frames at 10 &#181;m/step toward the blueshift direction to collect the hyperspectral image stack.</li>
		<li>To convert the frame numbers to wavenumbers, perform a linear regression using the symmetric (2,913 cm-1) and asymmetric (2,994 cm-1) C-H stretching from DMSO and their corresponding frame numbers40.</li>
		<li>Use the XPM signal for measuring the spectral focusing intensity profile. Half-close the diaphragm on the condenser and move the focus to a blank coverslip. Collect the same number of steps as for hyperspectral SRS. To measure the CARS nonresonant background, focus on the glass coverslip and collect the same number of steps for the hyperspectral CARS measurements.</li>
	</ol>
	</li>
	<li>Optimizing the signal-to-noise ratio (SNR) of images
	<ol>
		<li>Prepare a chemical sample for system alignment. Follow the procedure described in step 3.1 for sample preparation. 
		NOTE: DMSO is a good choice because it is a common lab chemical with strong Raman signals and well-separated C-H symmetric and asymmetric peaks.</li>
		<li>Place the sample on the microscope stage, and add water or immersion oil if needed for the objective lens or condenser. Properly move the edge of the DMSO droplet in the field of view and adjust the objective lens for the best focus. Center the condenser using the K&#246;hler illumination method41. Fully open the diaphragm on the condenser.</li>
		<li>Tune the pump beam wavelength to 800 nm (1,045 nm Stokes) to target the 2,913 cm-1 CH3 peak. Set the power of both the pump and Stokes beam to ~30 mW before the microscope by adjusting the half-wave plate (~10 mW power at the sample plane).</li>
		<li>For SRS, set the lock-in amplifier gain to ~10 with a time constant of 7 &#181;s (when using a 10 &#181;s pixel dwell time). Ensure that the time constant is smaller than the pixel dwell time. Use Demod R for the AUX output for SRS signals.</li>
		<li>For CARS, send the PMT output to the preamplifier and current-voltage converter. Use the DAQ to acquire the output from the converter.</li>
		<li>Set the image acquisition parameters in the acquisition software. Use a pixel number of 200 x 200 with a scanning size of ~100 x 100 &#181;m2. Make sure the image contains both the DMSO droplet and an empty area.</li>
		<li>Scan the sample and check the image on the computer screen. Scan the motorized delay stage in the Stokes/pump beam while monitoring the real-time images. Scan over the delay until the signal is maximized.</li>
		<li>Move the DMSO droplet to cover the whole field of view and check if the DC signal maximum is centered in the image (signal is pump beam-dependent). Adjust either the position of the pump beam via a mirror or the voltage offset in the imaging software.</li>
		<li>After DC optimization, adjust the Stokes beam mirrors until the AC signal is maximized by adjusting the threshold value to display ~50% saturation. Check whether the saturation is centered in the image. If not, fine-tune the mirrors only in the Stokes beam. Monitor the signal during alignment as real-time feedback on the quality of the alignment.</li>
		<li>To determine the SNR, select a small region of the DMSO image and measure the mean value. For the noise, select a small area in the empty region of the image and determine both the average mean value and the standard deviation. Subtract the noise mean value from the signal mean value and divide the results by the standard deviation of the empty region.</li>
		<li>If the calculated SNR is not high enough (typically 800-1,000 for SRS and &#62;10,000 for CARS at a PMT voltage of 0.4 V with this power combination), check and reoptimize the beam overlap, beam sizes, and delay stage position, fine-tune the AOM, and change the function generator modulation frequency until the expected SNR is obtained.</li>
	</ol>
	</li>
</ol>
2. Image analysis and data processing
<ol>
	<li>SNR analysis
	<ol>
		<li>Open the ImageJ software. To import the saved DMSO sample .txt file, click File | Import | Text Image | Open.</li>
		<li>Once the image is imported, press CTRL+shift+C to bring up the brightness and contrast function (B&#38;C). To find the maximum sample signal, press the auto button in the B&#38;C until a region of the DMSO sample appears saturated.</li>
		<li>Click the oval selection tool on the ImageJ interface and highlight a small area of the saturated DMSO region. Once highlighted, press M to measure the mean and standard deviation of the selected area.</li>
		<li>For measuring the background, adjust the bars in the B&#38;C function until the signal of the empty region can be observed. Click the oval selection and highlight a region of the background the same size as for step 2.1.3. Make sure the region selected does not contain DMSO. Press M to measure the statistics of the selected area.</li>
		<li>Calculate the SNR according to step 1.6.10.</li>
	</ol>
	</li>
	<li>Processing hyperspectral CRS images
	<ol>
		<li>Import the .txt file according to step 2.1.1. Once imported, click on Image | Stacks | Tools | Montage to Stack... to convert the file into an image stack.</li>
		<li>Scroll through the montage until the first DMSO peak is visible. Select a region on the DMSO and click on Image | Stack | Plot Z-axis Profile to plot the intensity versus frame number spectrum. To extract the raw spectral data, click on list and copy the profile data.</li>
		<li>To convert the recovered spectrum into wavenumber units, perform a linear regression as outlined in step 1.5.5.</li>
	</ol>
	</li>
	<li>Fitting for measuring spectral resolution 
	NOTE: Lorentzian functions are used to fit the SRS and CARS spectra28.
	<ol>
		<li>Open the fitting software, then copy and paste the linear regression data into the program. For fitting the SRS data, highlight the data and then plot the data as a scatter plot.</li>
		<li>Pull up the scatter plot. Click on Analysis | Peaks and Baseline | Multiple Peak Fit | Open Dialog to bring up the peak analyzer. When pulled up, check that the input is the current plot and change the peak function to Lorentzian (Lorentz).</li>
		<li>Double-click on each of the two DMSO peaks on the graph to highlight the regions to be fitted. Next, click on Open NLfit to bring up the fitting window. Click on the Fit until converged button, and then OK, to see a tabulated summary of the fitting coefficients (see Eq (3)). 
		NOTE: The below equation shows the Lorentzian function format in the software. A1/2 are the amplitudes of the fitting peaks, w1/2 are the widths of the fitted peaks, and the x01/02 values are the centers of the fitted peaks. The independent variable is x and the dependent variable is y. 
		<img alt="Equation 1" src="/files/ftp_upload/63677/63677eq3v2.png" style="margin: auto;" /> &#160; &#160; (3)</li>
		<li>For CARS spectral fitting, click on Analysis | Fitting | Nonlinear curve fit | Open Dialog. Select category: new to define a new function for CARS. Use a two-peak CARS fitting function defined below (see Eq (4)) for CARS spectral fitting. 
		<img alt="Equation 1" src="/files/ftp_upload/63677/63677eq4v2.png" style="margin: auto;" /> (4)</li>
	</ol>
	</li>
	<li>Determining the spatial resolution 
	NOTE: Before this step, it is important to know the conversion between the pixel size at a specific magnification, pixel number, and the step size in &#181;m. This can be performed by using a sample of a known diameter that is larger than the expected imaging resolution, measuring its line profile, and fitting a Gaussian function to determine the full width at half maximum (FWHM) value. Resolution targets or uniform samples such as polymeric beads can be used.
	<ol>
		<li>Acquire an image of cells or polymer particles less than 200 nm in diameter.</li>
		<li>Use ImageJ to draw a line across the smallest particle in the image.</li>
		<li>Press K to plot the intensity profile.</li>
		<li>Click list from the pop-up window and copy the information to fitting software.</li>
		<li>Plot the profile in fitting software and use Gaussian fitting (click on Analysis | Fitting | Nonlinear curve fit | Open Dialog | Category: Basic Functions; Function: Gauss).</li>
		<li>Read the peak width after fitting. Use the pixel to size conversion to obtain the actual resolution of the microscope.</li>
	</ol>
	</li>
</ol>
3. Preparation of samples for hyperspectral CRS imaging
<ol>
	<li>Preparation of imaging slides and chemical samples
	<ol>
		<li>Place a piece of double-sided tape onto a coverslip, and cut out a small rectangular shape of the tape from the middle of the placed tape to create an open area for the sample to be placed.</li>
		<li>Pipette 1-2 &#181;L of pure DMSO and dispense the droplet at the center of the vacancy.</li>
		<li>Carefully place the top coverslip and gently press the edges of the coverslips to seal the chamber while making sure the DMSO sample does not contact the edges of the tape.</li>
		<li>For sensitivity experiments, prepare serial dilutions of DMSO in deuterium oxide (D2O) to give a concentration range of 50%-0%. Take 1-2 &#181;L of each solution and prepare pressed samples as described above.</li>
	</ol>
	</li>
	<li>Cell preparation
	<ol>
		<li>Seed the cells in a 35 mm glass-bottom dish (or larger) in Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.</li>
		<li>Incubate the cells in an incubation chamber at 37 &#176;C with a 5% CO2 atmosphere&#160;overnight or longer until ~50%-80% confluency is achieved.</li>
		<li>Image the live cells directly or fix the cells with a 10% formalin solution for imaging.</li>
	</ol>
	</li>
</ol>

The authors declare no conflicts of interest.

The protocol presented here describes the construction of a multimodal CRS microscope and the direct comparison between CARS and SRS imaging. For the microscope construction, the critical steps are spatial and temporal beam overlapping and beam size optimization. It is recommended to use a standard sample such as DMSO before the biological imaging for optimizing SNR and calibrating Raman shifts. Direct comparison between CARS and SRS images reveals that CARS has a better spatial resolution, while SRS gives better spectral resolution and less convoluted chemical contrasts. Both CARS and SRS have similar detection limits.
CARS and SRS imaging use high-energy pulse lasers for excitation. This allows the platform to integrate other nonlinear optical imaging modalities such as multiphoton excitation fluorescence, harmonic generation, and transient absorption for additional chemical contrasts28,39.
CARS and SRS have been extensively used to study lipid composition with high chemical selectivity. However, the technologies are not limited to quantifying lipids. SRS has been applied to map drug distribution42, protein synthesis43, and DNA44. CARS and SRS have also been applied to image pharmaceutical ingredients and excipients in tablets45,46,47,48. Hyperspectral CARS and SRS have found applications in cancer diagnosis49, cardiovascular disease evaluation50, and neural imaging51. They can also be applied for COVID-19 studies52. Broadband CARS, which can cover spectral windows as broad as 3,000 cm-1, can elucidate rich chemical structures in biological samples53. However, due to the slow readout rate of the CCD, the pixel dwell time is on the level of milliseconds, much slower than the microsecond pixel dwell time for SRS microscopy34. Hyperspectral SRS microscopy currently has a typical bandwidth of 200-300 cm-1, limited by the laser bandwidth and the lack of lock-in-integrated array detectors34. Fourier transform SRS microscopy is an alternative way to potentially broaden the SRS spectral coverage35.
Although CARS and SRS provide rich chemical information without the need for labeling, the chemical selectivity lies in chemical bonds, making it difficult to distinguish specific proteins. Raman tags have shown the potential to enhance the chemical selectivity of CARS and SRS54,55. However, coherent Raman imaging still has much lower sensitivity compared to fluorescence detection. Surface enhancement was used for spontaneous Raman scattering spectroscopy to improve the signal levels56. It was also applied to CARS and SRS for signal amplification57,58,59. Although the enhancement factor is not as high as spontaneous Raman scattering, surface-enhanced CARS and SRS microscopy still show the potential to detect single molecules59,60. Nevertheless, the use of metal particles or surfaces deprives the advantage of the label-free approach. Improving the sensitivity of coherent Raman microscopy without using metal surfaces would greatly expand the application of the technology in biological science.

The Raman scattering phenomenon was first observed in 1928 by C. V. Raman1. When an incident photon is interacting with a sample, an inelastic scattering event can spontaneously occur, in which the energy change of the photon matches a vibrational transition of the analyzed chemical species. This process does not require the use of a chemical tag, making it a versatile, label-free tool for chemical analysis while minimizing sample perturbation. Despite its advantages, spontaneous Raman scattering suffers from a low scattering cross-section (typically 1011 lower than the infrared [IR] absorption cross-section), which necessitates long acquisition times for analysis2. Thus, the quest for increasing the sensitivity of the Raman scattering process is essential in pushing Raman technologies for real-time imaging.
One effective way to greatly enhance the sensitivity of Raman scattering is through coherent Raman scattering (CRS) processes, for which two laser pulses are typically used to excite molecular vibrational transitions3,4. When the photon energy difference between the two lasers matches the vibrational modes of sample molecules, strong Raman signals will be generated. The two most commonly used CRS processes for imaging are coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)5. Over the past two decades, technological developments have advanced CARS and SRS microscopy techniques to become powerful tools for label-free quantification and elucidation of chemical changes in biological samples.
Chemical imaging by CARS microscopy can be dated to 1982 when laser scanning was first applied to acquire CARS images, demonstrated by Duncan et al6. The modernization of CARS microscopy was greatly accelerated after the wide applications of laser scanning multiphoton fluorescence microscopy7. Early work from the Xie group using high repetition rate lasers has transitioned CARS to be a high-speed, label-free, chemical imaging platform for the characterization of molecules in biological samples8,9,10. One of the major issues for CARS imaging is the presence of a nonresonant background, which reduces the image contrast and distorts the Raman spectrum. Many efforts have been made to either reduce the nonresonant background11,12,13,14,15 or to extract resonant Raman signals from the CARS spectra16,17. Another advancement that has greatly advanced the field is hyperspectral CARS imaging, which allows for spectral mapping at each image pixel with improved chemical selectivity18,19,20,21.
Stimulated Raman scattering (SRS) is a younger imaging technology than CARS, although it was discovered earlier22. In 2007, SRS microscopy was reported using a low repetition rate laser source23. Soon, several groups demonstrated high-speed SRS imaging using high repetition rate lasers24,25,26. One of the major advantages of SRS microscopy over CARS is the absence of the nonresonant background27, although other backgrounds such as cross-phase modulation (XPM), transient absorption (TA), two-photon absorption (TPA), and photothermal (PT) effect, may occur with SRS28. In addition, the SRS signal and sample concentration have linear relationships, unlike CARS, which has a quadratic signal-concentration dependence29. This simplifies chemical quantification and spectral unmixing. Multicolor and hyperspectral SRS has evolved in different forms30,31,32,33,34,35,36, with spectral focusing being one of the most popular approaches for chemical imaging37,38.
Both CARS and SRS require the focusing of the pump and Stokes laser beams onto the sample to match the vibrational transition of the molecules for signal excitation. CARS and SRS microscopes also share a lot in common. However, the physics underlying these two processes, and signal detections involved in these microscopy technologies have disparities3,39. CARS is a parametric process that does not have net photon-molecule energy coupling3. SRS, however, is a nonparametric process, and contributes to energy transfer between photons and molecular systems27. In CARS, a new signal at anti-Stokes frequency is generated, while SRS manifests as the energy transfer between the pump and Stokes laser beams.
CARS signal satisfies Eq (1)28.
<img alt="Equation 1" src="/files/ftp_upload/63677/63677eq1v2.png" style="margin: auto;" /> &#160; &#160; (1)
Meanwhile, SRS signal can be written as Eq (2)28.
<img alt="Equation 1" src="/files/ftp_upload/63677/63677eq2v2.png" style="margin: auto;" />&#160; &#160; (2)
Here, Ip, Is, ICARS, and &#916;ISRS are the intensities of the pump beam, Stokes beam, CARS signal, and SRS signals, respectively.&#160;&#967;(3) is the third-order nonlinear optical susceptibility of the sample, and is a complex value composed of real and imaginary parts.
These equations express the spectral profiles and signal-concentration dependence of CARS and SRS. Differences in physics result in disparate detection schemes for these two microscopy technologies. Signal detection in CARS usually involves spectral separation of newly generated photons and detection using a photomultiplier tube (PMT) or charge-coupled device (CCD); for SRS, the energy exchange between the pump and Stokes beams is usually measured by high-speed intensity modulation using an optical modulator and demodulation using a photodiode (PD) paired with a lock-in amplifier.
Although many technological developments and applications have been published in recent years in both CARS and SRS fields, no systematic comparisons of the two CRS techniques have been performed on the same platform, especially for hyperspectral CARS and SRS microscopy. Direct comparisons in sensitivity, spatial resolution, spectral resolution, and chemical separation capabilities would allow biologists to select the best modality for chemical quantification. In this protocol, detailed steps to construct a multimodal imaging platform with both hyperspectral CARS and SRS modalities based on a femtosecond laser system and spectral focusing are provided. The two techniques have been compared in the forward direction for spectral resolution, detection sensitivity, spatial resolution, and imaging contrasts of cells.

<table><tbody><tr><td>2D galvo scanner set</td><td>Thorlabs</td><td>GVS002</td><td></td></tr><tr><td>Acousto-optic modulator</td><td>Isomet</td><td>M1205-P80L-0.5</td><td></td></tr><tr><td>AOM driver</td><td>Isomet</td><td>532B-2</td><td></td></tr><tr><td>Data acquisition card</td><td>National Instruments</td><td>PCle 6363</td><td>Custom ordered filter (980 sp)</td></tr><tr><td>Delay stage</td><td>Zaber</td><td>X-LSM050A</td><td></td></tr><tr><td>Deuterium oxide</td><td>Millipore Sigma</td><td>151882-100G</td><td></td></tr><tr><td>Dichroic mirror for beam combination</td><td>Thorlabs</td><td>DMLP1000</td><td></td></tr><tr><td>Dichroic mirror for signal separation</td><td>Semrock</td><td>FF776-Di01-25x36</td><td></td></tr><tr><td>DMSO</td><td>MiliporeSigma</td><td>200-664-3</td><td></td></tr><tr><td>MIA PaCa 2 Cells</td><td>ATCC</td><td>CRL-1420</td><td></td></tr><tr><td>Femtosecond laser system</td><td>Spectral Physics</td><td>InSightX3+</td><td></td></tr><tr><td>Filter for CARS</td><td>Chroma</td><td>AT655/30m</td><td></td></tr><tr><td>Filter for SRS</td><td>Chroma</td><td>ET980sp</td><td></td></tr><tr><td>Function generator</td><td>Rigol</td><td>DG1022Z</td><td></td></tr><tr><td>Glass rods</td><td>Lattice Electro Optics</td><td>SF-57</td><td></td></tr><tr><td>Half-wave plate</td><td>Newport</td><td>10RP02-51; 10RP02-46</td><td></td></tr><tr><td>LabVIEW 2020</td><td>National Instruments</td><td></td><td>This is the image acquisition software</td></tr><tr><td>Lock-in amplifier</td><td>Zurich Instrument</td><td>HF2LI</td><td></td></tr><tr><td>Microscope housing</td><td>Olympus</td><td>BX51W1</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>UPLSAPO60XW</td><td></td></tr><tr><td>Origin Pro 2019b</td><td>OriginLab Corporation</td><td></td><td>This is the spectral fitting software</td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>TBS2204B</td><td></td></tr><tr><td>Photodiode</td><td>Hamamatsu</td><td>S3994-01</td><td></td></tr><tr><td>PMT detector</td><td>Hamamatsu</td><td>H7422P-40</td><td></td></tr><tr><td>PMT voltage amplifier</td><td>Advanced Research Instrument Corp.</td><td>PMT4V3</td><td></td></tr><tr><td>Polarizing beamsplitter cube</td><td>Thorlabs</td><td>PBS255</td><td></td></tr><tr><td>Terminal block</td><td>National Instruments</td><td>BNC-2110</td><td></td></tr></tbody></table>

direct comparison of hyperspectral stimulated raman scattering and coherent anti-stokes raman scattering microscopy for chemical imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

Comparisons of the spectral resolution 
Figure 2 compares the spectral resolution of hyperspectral SRS (Figure 2A) and CARS (Figure 2B) microscopy using a DMSO sample. For the SRS spectrum, two Lorentzian functions (see protocol step 2.3) were applied to fit the spectrum, and a resolution of 14.6 cm-1 was obtained using the 2,913 cm-1 peak. For CARS, a two-peak-fitting function with a Gaussian background (see protocol step 2.3) was utilized for fitting, which gave the spectral resolution of 17.1 cm-1. These results show that, in the same measurement condition, SRS has a better spectral resolution than CARS. The reduced spectral resolution in CARS is mainly contributed by the involvement of the nonresonant background. In addition, it was found that the symmetric (2,913 cm-1) and asymmetric (2,995 cm-1) peak ratios were very different for SRS and CARS. This is due to the different signal correlations with the third-order nonlinear optical susceptibility, as described in equations (1) and (2). With the quadratic dependence of CARS, the intensity difference between the two peaks is amplified. The symmetric line shapes of the SRS peaks and the asymmetric line shapes of the CARS peaks can be observed in the spectrum. The asymmetry in the CARS signal is mainly due to the presence of the nonresonant background interference. The CARS spectral peaks appear slightly red-shifted (1-2 cm-1) to the SRS peaks. This also arises from the nonresonant background interference with resonant peaks.
Comparisons of the detection sensitivity 
Figure 3 compares the detection sensitivity of hyperspectral SRS and CARS microscopy. The SNR of the DMSO SRS signals (2,913 cm-1) as the function of DMSO concentration in D2O at high concentrations are plotted first (1%-50%, Figure 3A). The results show a linear relationship, satisfying equation (2). Figure 3B plots the DMSO spectra at 0.1% and 0.01% concentrations, in which the 2,913 cm-1 peak can be resolved in the former but not in the latter, indicating the detection limit is between 0.1% and 0.01% DMSO. Using the limit of blank criteria, we estimated that the SRS detection limit is 0.021% DMSO. Figure 3C plots the CARS SNR as the function of DMSO concentration (1%-50%), showing a quadratic dependence in agreement with equation (1). The phase-retrieved CARS spectra are shown in Figure 3D for the 0.1% and 0.01% DMSO. To achieve these spectra, a spectral phase-retrieval method based on Kramers-Kronig relations was used and additional background removal was performed16. Similar to the SRS spectra, the DMSO 2,913 cm-1 peak can be clearly resolved for the 0.1% DMSO but not the 0.01%, indicating a detection limit between these two concentrations. Using the limit of blank criteria, we estimated that the SRS detection limit is 0.015% DMSO. The 0.02% DMSO corresponds to 2.8 mM. Therefore, the detection limit of the hyperspectral CRS microscope used here is ~2.1-2.8 mM DMSO.
Comparisons of the spatial resolution 
Figure 4 compares the resolution of a small cellular feature detected in SRS (Figure 4A) and CARS (Figure 4B) images. The intensity profiles from the same line are displayed and fit using a Gaussian function to determine FWHM values for resolution comparison. The SRS signal gave a resolution of 398.6 nm (Figure 4C), while the CARS signal gave a resolution of 330.3 nm (Figure 4D). The resolution of CARS was ~1.2x better than that of the SRS. The reason for the resolution difference also lies in equations (1) and (2). Both pump and Stokes beams have a Gaussian point spread function at the focus. The signal of CARS is then proportional to the multiplication of three Gaussian functions, which roughly reduces the width by a factor of &#8730;3. Similarly, for SRS, the width is reduced by a factor of &#8730;2. Therefore, the resolution of CARS was &#8730;3/&#8730;2 = 1.2 times better than that of the SRS.
Comparisons of the images of cells 
Figure 5 compares SRS and CARS images from MIA PaCa-2 cells at different optical delay positions. Figure 5A shows the SRS images at the optical delay that gave the strongest signal. In this image, lipid droplets (LDs), endoplasmic reticulum (ER), and nucleus (NU) can be detected, with LDs having the strongest signals shown as bright dots. Figure 5B shows the CARS channel image at the same optical delay, having much-reduced contrasts for LDs. The major reasons for this contrast difference are the presence of the nonresonant background and the red-shift of the same Raman peak in CARS spectra. At this optical delay, the generated signal has a large contribution from the nonresonant background of water. To enhance the lipid contrast in CARS, the optical delay was tuned to a red-shifted value. The red-shift improved the lipid contrasts by concentrating more energy to the 2,850 cm-1 for both SRS (Figure 5C) and CARS (Figure 5D), although the overall signal level was reduced. For CARS, a similar contrast of LDs as SRS was achieved by a ~98 cm-1 red-shift in spectral focusing (Figure 5D), though a background higher than that in the SRS image was still observed. At this optical delay, the SRS image shows much less protein and nucleic acid contents but strong lipid contents in LDs, ER, and cell membranes (Figure 5C).
CARS is a parametric process while SRS is nonparametric. Such a difference also contributes to contrast differences in the two modalities. The parametric CARS signals are determined by the interference of CARS signals from different layers close to the laser focus, which might show negative contrasts as indicated by arrows in Figure 5B and Figure 5D (also in Figure 4B). Such signal-interference-induced negative contrasts are absent in the SRS images. The negative contrast in CARS might provide information about the axial position of the target of interest.
The SRS signals have a linear relationship with the molecular concentration, while the CARS signals satisfy a near-quadratic concentration-dependence. Therefore, the CH2-rich LDs show a much stronger signal than the ER and the cell membranes in the CARS image than in the SRS image (Figure 5E,F). The SRS spectra can be extracted from hyperspectral images. Figure 5G shows the typical SRS spectra from LDs, ER, cytosol (CY), and NU. Both the intensity and spectral shape are different for different cellular compartments. LD shows a much stronger signal at 2,850 cm-1 than other organelles. As for CARS, similar spectra, though different in shapes, can be obtained. The raw CARS spectra show a small red-shift compared to the corresponding SRS spectra. Spectral phase-retrieval can be further used to extract the Raman responses using the CARS spectra.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63677/63677fig01.jpg" /> 
Figure 1: A schematic of the hyperspectral CARS/SRS microscope. Abbreviations: CARS = coherent anti-Stokes Raman scattering; SRS = stimulated Raman scattering; PBS = polarization beam splitter; PD = photodiode; PMT = photomultiplier tube; AOM = acousto-optic modulator. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63677/63677fig01large.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/63677/63677fig02.jpg" /> 
Figure 2: DMSO spectra. (A) SRS and (B) CARS spectra of DMSO. Dots are experimental data; curves are spectral fitting results. Abbreviations: CARS = coherent anti-Stokes Raman scattering; SRS = stimulated Raman scattering; DMSO = dimethyl sulfoxide; w = spectral resolution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63677/63677fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63677/63677fig03.jpg" /> 
Figure 3: Signal-to-noise ratios and spectra of DMSO. (A) Signal-to-noise ratio of the DMSO symmetric peak at 2,913 cm-1 as a function of concentration in D2O measured by SRS. The dots are experimental data; the line is the linear fitting result. (B) The SRS spectra of 0.1% and 0.01% DMSO in D2O. (C) Signal-to-noise ratio of the DMSO symmetric peak at 2,913 cm-1 as a function of concentration in D2O measured by CARS. The dots are experimental data; the curve is the second-degree polynomial fitting result. (D) The CARS spectra of 0.1% and 0.01% DMSO in D2O. Abbreviations: CARS = coherent anti-Stokes Raman scattering; SRS = stimulated Raman scattering; DMSO = dimethyl sulfoxide; SNR = signal-to-noise ratio; D2O = deuterium oxide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63677/63677fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63677/63677fig04.jpg" /> 
Figure 4: SRS and CARS images and intensity profiles of an MIA PaCa-2 cell. (A) An SRS image of an MIA PaCa-2 cell. (B) A CARS image of an MIA PaCa-2 cell at the same field-of-view as panel A. (C) Intensity profile of SRS along the yellow line in panel A. (D) Intensity profile of CARS along the yellow line in panel B. Dots are experimental data; curves are Gaussian function fitting results. Scale bars = 5 &#181;m. Abbreviations: CARS = coherent anti-Stokes Raman scattering; SRS = stimulated Raman scattering; w = resolution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63677/63677fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63677/63677fig05v2.jpg" /> 
Figure 5: Images and intensity profile of MIA PaCa-2 cells. (A) An SRS image of MIA PaCa-2 cells at the optimized time delay for SRS intensity. (B) A CARS image at the same delay as in panel A. (C) An SRS image at the 98 cm-1 red-shifted delays as in panel A. (D) A CARS image at the same optical delay as in panel C. (E,F) The SRS and CARS intensity profiles plotted along the dotted lines in panels A and D. (G) Typical SRS spectra from the lipid droplets, endoplasmic reticulum, cytosol, and nucleus. (H) Typical CARS spectra of the four cellular compositions. The green and red dotted lines are delay positions for panels A/B and C/D, respectively. Scale bars = 10 &#181;m. Abbreviations: CARS = coherent anti-Stokes Raman scattering; SRS = stimulated Raman scattering; LD = lipid droplets; ER = endoplasmic reticulum; CY = cytosol; NU = nucleus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63677/63677fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File: Lab-written software based on LabVIEW having a simultaneous multichannel display for real-time viewing and saving images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63677/Laser scanning imaging software.vi" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging at JoVE.com

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

direct-comparison-hyperspectral-stimulated-raman-scattering-coherent

Nanyang Technological University

Research

JoVE Journal

Chemistry

3.8K Views.  Purdue University. Dit protocol vergelijkt de twee meest gebruikte hyperspectrale coherente Raman-verstrooiingstechnieken, waaronder de coherente anti-Stokes Raman-verstrooiing en de gestimuleerde Raman-verstrooiingsprocessen en dit stelt de biologen in staat om de beste beeldvormingsmodaliteit voor hun biologische toepassingen te kiezen. Met behulp van de intrinsieke trillingshandtekeningen is coherente Raman-spectroscopie in staat om chemische informatie en biologische monsters in kaart te brengen zonder dat ...

Video: Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Engineering

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 ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Biology

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Bioengineering

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

Neuroscience

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

Medicine

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

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