This protocol will demonstrate implementation and optimization of spectral focusing SRS microscopy and how it can be used for real-time stimulated Raman histology applications. The main advantage of this technique is the speed at which samples can be processed once the instrumentation is properly aligned, as SRS does not require tissue processing and staining. This protocol is applicable to other SRS applications, not just limited to histology.
Such applications include small molecules, drugs, cells, and tissues. Begin by installing a pair of achromatic lenses for the pump beam to magnify the laser beam size by twofold as a starting point. Place a 100 and 200 millimeter lens at roughly 300 millimeters from each other.
Then align the pump beam through the center of both lenses. Next place a mirror after the second lens to send the beam towards a wall more than one meter away. Trace the beam from the mirror to the wall with an IR card and check if the beam changes in size.
Collimate the beam if the beam changes in size as a function of distance. Combine the two laser beams by installing a dichroic mirror with several steering mirrors for adjustment. Optimize spatial overlap of the pump and Stokes by monitoring beams after the dichroic mirror at two different positions around one meter apart.
Iteratively adjust the steering mirror followed by the dichroic mirror to align the Stokes beam with the pump beam. Ensure that the combined beams are sent to the center of the scan mirrors of the laser scanning microscope by adjusting a pair of steering mirrors when the scan mirrors are in the parked position. After the condenser, use another pair of lenses with focal lengths of 100 millimeters and 30 millimeters, respectively, to relay the transmitted beam onto the photodiode, ensuring both beams are contained within the photodiode.
Then install two low pass filters to block out the modulated Stokes beam. Place a beam sampler in the Stokes beam path to pick up 10%of the beam and send it to a fast photodiode to detect the 80 megahertz pulse train. Take one of the outputs of the fanout buffer and filter it with a band pass filter to obtain a 20 megahertz sinusoidal wave.
Then use an RF attenuator to adjust the output peak to peak voltage to approximately 500 millivolts. Send the resulting output to a phase shifter, which allows fine adjustment of the RF phase with a voltage source. Send this output to an RF power amplifier and connect the output of the amplifier to the EOM.
After unblocking the Stokes beam, optimize the modulation depth of the first EOM by placing a photodiode in the beam path. Adjust the EOM voltage and quarter wave plate until the modulation depth appears satisfactory. Place a photodiode after the dichroic mirror to detect the laser beam.
Block the Stokes beam first, then zoom in on one of the pump pulse peaks on the oscilloscope. Place a vertical cursor to mark the temporal position of this peak with the oscilloscope. Now block the pump beam, and unblock the Stokes beam.
Translate the delay stage to temporarily match the peak positions on the oscilloscope to the marked position in the previous step. By calculating the delay distance required to match the two beams followed by elongating the beam path of the faster beam or shortening the beam path of the slower beam. Next prepare a microscope slide sample with DMSO and double-sided tape as a spacer to hold the sample between the slide and a coverslip.
Place the sample on the microscope with the coverslip side facing the microscope objective and observe the sample from the I piece under brightfield illumination. Find the focus of the sample at both the top and bottom layer of air bubbles at the glass tape interface, then move the focus to be in between the two layers of the tape. Next set the tunable beam output to 798 nanometers.
Based on the optical throughput of the condenser, adjust the optical power to be approximately 40 milliwatts each for the pump and Stokes beams at the objective focus. Then open scan image in MATLAB and click on the button labeled focus to start scanning. Adjust the steering mirror before the galvo scanner to center the DC signal on the channel one display.
Move the motorized delay stage and closely observe the lock-in output shown on the channel two display. Finally adjust the time delay to maximize the AC signal intensity. Adjust the dichroic mirror to center the SRS signal on the AC channel.
Then adjust the phase of the lock-in amplifier to maximize the signal. After mounting the sample slide onto the microscope and adjusting the power of both beams to approximately 40 milliwatts each at sample focus as demonstrated earlier. Open scan image from MATLAB.
Then find the maximal SRS signal by scanning through the delay stage corresponding to the 2, 913 inverse centimeter Raman peak of DMSO. Acquire an SRS image corresponding to the 2, 913 inverse centimeter Raman peak of DMSO. Open the image in ImageJ and select a small area in the center of the frame.
Use the measure function to calculate the mean and standard deviation of values in the selected area. For frequency access calibration, save a hyperspectral scan with the delay stage range covering the 2, 913 and 2, 994 inverse centimeter Raman peaks of DMSO. Then save the stage positions corresponding to the hyperspectral data set.
Perform linear regression for the stage positions and Raman shifts at 2, 913 and 2, 994 inverse centimeters. Using the linear regression equation, convert the delay positions to the corresponding Raman frequencies. For calibration of spectral resolution, estimate the stage position based on the previous position with the increased optical path length due to the insertion of rods.
Realign the beams spatial overlap because of the slight deviation of the beam when glass rods are added. Install a polarizing beam splitter, a quarter wave plate, and a second EOM into the soaks beam path after the first EOM. Unplug the signal input to the second EOM, then plug in the signal input to the first EOM, and turn it on.
Modulate the Stokes beam at 20 megahertz by sending the beam through the first EOM. Adjust the tilt and position of the first EOM to ensure that the beam is centered through the EOM crystal. Prepare a tissue slide sample with double-sided tape, microscope slide and cover glass.
Place the sample on the microscope with the coverslip side facing the microscope objective. For epi-mode SRS imaging, install a half-wave plate before the beam enters the microscope to change the polarization of the beam going into the microscope. Place a polarizing beam splitter above the objective to allow the depolarized back reflected beam to reach the detector.
Next, use a 75 millimeter achromate lens and a 30 millimeter aspheric lens to relay the back scattered photons from the back aperture of the objective to the photo detector. Mount the detector to collect the back scattered light directed by the polarizing beam splitter. Then install a filter to block out the modulated beam from entering the detector.
Varying the rod lengths affect spectral resolution. When no glass chirping rods are used, the two Raman peaks from DMSO are not resolved at all. An increase in the number of glass chirping rods starts to resolve the two peaks at a satisfactory point.
Matched chirping resolves both peaks and can be used to calibrate stage positions to frequency. Two time delayed pulse trains of orthogonal polarization used to image DMSO spectra show the time delay between the SRS excitations with acceptable modulation depth and temporal separation. In contrast, a poor two color SRS phase shift, gives rise to inverted or negative peaks.
Real-time two color SRS imaging at 2, 850 and 2, 930 inverse centimeters of ex vivo mouse brain tissue are shown here. The raw lipid and protein images were color coded to produce a single image depicting lipid and protein contributions. False hematoxylin and eosin recoloring, were also performed to mimic hematoxylin and eosin staining for pathological applications.
Spatial temporal overlap and spatial resolution optimization are the most important steps for the spatial focusing approach of SRS. This method is generally applicable for other pump probe microscopy experiments such as transient absorption microscopy. The method also allows for imaging of non-fluorescent molecules in cells and tissue.
The method presented here speeds up the image acquisition time for future translation of stimulated Raman histology in the clinic.
