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.
