Two-Color Stimulated Raman Scattering Imaging of Mouse Brain Tissue

Take a mouse brain slice inside a chamber on a slide and place it under a microscope with a stimulated Raman scattering, or SRS, imaging system.

Focus two synchronized laser beams, known as the pump and Stokes beams, onto the tissue.

The energy difference between these beams matches the vibrational frequency of specific molecules, inducing molecular vibrations.

Excite the tissue lipids and proteins, having unique molecular vibrations, using distinct frequency pairs of the synchronized beams.

The excitation facilitates energy transfer between the beams, reducing the pump beam intensity while increasing the Stokes beam intensity.

The relative intensity change is detected as an SRS signal that helps identify the molecules.

A photodetector collects the combined SRS signals from the sample. 

Assign the lipid and protein signals separate colors, creating a two-colored image, to identify the proteins and lipids in the brain tissue.

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 8 megahertz pulse train. Take one of the outputs of the fanout buffer and filter it with a bandpass filter to obtain the 20 megahertz sinusoidal wave. Then, use an RF attenuator to adjust the input/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 eyepiece 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 input/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 1 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 2913 inverse centimeter Raman peak of DMSO.

Acquire an SRS image corresponding to the 2913 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 axis 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 beam 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 stokes 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 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 slice 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 achromat lens and a millimeters aspheric lens to relay the backscattered photons from the back aperture of the objective to the photodetector. Mount the detector to collect the backscattered light directed by the polarizing beam splitter. Then install a filter to block out the modulated beam from entering the detector.