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 o...

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 spectra...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 Gaus...

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 ...

This protocol compares the two most widely-used hyperspectral coherent Raman scattering techniques including the coherent anti-Stokes Raman scattering and the stimulated Raman scattering processes and this allows the biologists to choose the best imaging modality for their biological applications. Using the intrinsic vibrational signatures, coherent Raman spectroscopy is able to map out chemical information and biological samples without the need for exogenous labeling. Coherent Raman microscopy has expanded our understanding of cellular metabolism, mapping, drug distributions, disease diagnostics, and quantifying chemical changes.This is a technique that requires sufficient training in both optics and spectroscopy. I would recommend reading a few review articles and being trained by an expert before using this technique. To begin, prepare the imaging slides by placing a piece of double-sided tape onto a cover slip and cut out a small rectangular shape of the tape from the middle of the tape to create an open area for the sample to be placed.Pipette 1 to 2 microliters of pure DMSO and dispense the droplet at the center of the vacancy. Carefully place the top cover slip and gently place the edges of the cover slips to seal the chamber while making sure the DMSO sample does not contact the edges of the tape. For sensitivity experiments, prepare serial dilutions of DMSO in deuterium oxide to give a concentration range of 50 to 0%Take 1 to 2 microliters of each solution and prepare pressed samples as demonstrated previously.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, then center the condenser using the Kohler illumination method and fully open the diaphragm on the condenser. Next, tune the pump beam wavelength to 800 nanometers to target the 2913 wavenumber CH3 peak and set the power of both the pump and the Stokes beam to approximately 30 milliwatts before the microscope by adjusting the half-waveplate.For SRS, set the lock-in amplifier gain to approximately 10 with a time constant of 7 microseconds, ensuring that the time constant is smaller than the pixel dwell time. Set the image acquisition parameters and the acquisition software using a pixel number of 200 by 200 with the scanning size of approximately 100-by-100-square micrometers, ensuring that the image contains both the DMSO droplet and an empty area, then scan the sample and check the image on the computer screen. Next, scan the motorized delay image in the Stokes pump beam while monitoring the real-time images and scan over the delay until the signal is maximized.Move the DMSO droplet to cover the whole field of view and check if the DC signal maximum is centered in the image as the signal is pump beam-dependent. Adjust either the position of the pump beam via a mirror or the voltage offset in the imaging software. After DC optimization, adjust the Stokes beam mirrors until the AC signal is maximized by adjusting the threshold value to display approximately 50%saturation while checking that the saturation is centered in the image.If not, fine-tune the mirrors only in the Stokes beam and monitor the signal during alignment as real-time feedback on the quality of the alignment. For SNR analysis, open the ImageJ software and import the saved DMSO sample text file by clicking on File, then Import, followed by the Text Image and Open options from the drop-down menu. Once the image is imported, press Control Shift C to bring up the Brightness and Contrast function, then press the Auto button in the Brightness and Contrast function until the region of the DMSO sample appears saturated to find the maximum sample signal.Next, click the Oval selection tool on the ImageJ interface and highlight a small area of the saturated DMSO region. After highlighting, press M to measure the mean and standard deviation of the selected area. For measuring the background, adjust the bars in the Brightness and Contrast function until the signal of the empty region can be observed, then click the Oval selection and highlight a region of the background, ensuring that the region selected does not contain DMSO.Afterward, press M to measure the statistics of the selected area. Next, calculate the SNR as demonstrated previously by measuring the noise mean value and signal mean value along with the standard deviation. To process the hyperspectral CRS images, import the text file by clicking on File, then Import, Text Image, and Open options from the drop-down menu.Once imported, click on Image, then Stacks, followed by Tools and Montage to Stack options to convert the file into an image stack, then scroll through the montage until the first DMSO peak is visible. Select a region on the DMSO and click on Image, followed by Stack and Plot Z-axis Profile options to plot the intensity versus frame number spectrum. Next, click on List and copy the profile data to extract the raw spectral data.To convert the recovered spectrum into frequency units, perform a linear regression using the symmetric and asymmetric CH stretching from DMSO and their corresponding frame numbers. The spectral resolution of the DMSO was measured using a hyperspectral SRS and CARS microscopy, which show a resolution of 14.6 and 17.1 wavenumber respectively, indicating that SRS has a better spectral resolution. The DMSO SRS spectra were obtained at 0.1 and 0.01%concentrations in which the peak at 2913 wavenumber can be resolved in the former, but not in the latter, indicating the detection limit is between 0.1 and 0.01%DMSO.The phase-retrieved CARS spectra show the DMSO 2913 wavenumber peak can be clearly resolved for the 0.1%DMSO, but not the 0.01%indicating a detection limit between these two concentrations. SRS and CARS intensity profiles of a MIA PaCA-2 cell demonstrated that the SRS signal gave a resolution of 398.6 nanometers, while the CARS signal gave 1.2 times better resolution of 330.3 nanometers. The SRS and CARS images from MIA PaCa-2 cells at different optical delay positions show the strongest signals with lipid droplets as bright dots for SRS, whereas CARS have much reduced contrasts.However, a 37 wavenumber red shift in spectral focusing improved the lipid contrasts for both SRS and CARS. The SRS spectra show a much stronger signal at 2850 wavenumber for lipid droplets than other organelles, whereas the CARS spectra show a small red shift. To optimize the coherent Raman scattering signal, first find the sample focus, then tune the optical delay, and finally, fine-tune the mirrors until a maximum is achieved.Other pump-probe technologies, such as transient absorption are inherently integrated into the CRS platform. This technology is very powerful for measuring the absorption kinetics of strong light absorbing non-fluorescent molecules. This technique allows researchers to view small molecules in a label-free manner with high chemical cell activity.It also allows researchers to view changes in lipid metabolism, intracellular dynamics, and drug distributions.

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3.6K visualizações.  Purdue University. Este protocolo compara as duas técnicas de dispersão raman hiperespectral mais utilizadas, incluindo a dispersão coerente anti-Stokes Raman e os processos estimulados de dispersão de Raman e isso permite que os biólogos escolham a melhor modalidade de imagem para suas aplicações biológicas. Usando as assinaturas vibracionais intrínsecas, a espectroscopia raman coerente é capaz de mapear informações químicas e amostras biológicas sem a necessidade de rotulagem exógena. A microsc...