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

In Vivo imaging of biological tissues with subcellular resolution and chemical specificity is a powerful tool to understand the dynamic processes involved in cellular metabolism, immune response, and tissue remodeling. With combined two-photon fluorescence and stimulated Raman scattering microscopy, critical biochemical and biophysical information of biological tissues can be acquired from multiple perspectives with subcellular resolution. Specifically, this dual-modal microscopy can be used to image cellular dynamics and interactions in mouse spinal cord to study the progression of neurodegenerative diseases and spinal cord injury.

To begin, turn on the titanium sapphire laser by switching the key switch from the standby to on position and wait for 30 minutes for the laser to warm up. Then switch on the OPO by clicking the start button on the OPO control panel and wait for 20 minutes for the laser to warm up. After the picosecond laser warms up, check the modulation depth of the Stokes beam by lowering the power of the Stokes beam to approximately 20 milliwatts, opening the laser shutter for the Stokes beam and placing a high-speed photo detector at the OPO output to detect the beam.

Next, connect the output port of the photodetector to the input port of an oscilloscope using a coaxial cable with a BNC connector to monitor the laser pulse. Then open the EOM control window in the OPO control software and adjust the EOM power and phase according to the pulse intensity diagram shown on the oscilloscope to achieve maximum modulation depth at 20 megahertz. In the OPO control software, open the pump laser shutter while stopping the Stokes output.

Next, click the set signal box to set the wavelength of the pump beam to 796 nanometers, then click the set OPO power box and enter 20 to set its power to the minimum for optical alignment. Next, place alignment plate P1 behind the polarizing beam splitter at about 10 centimeters and place plate P2 behind P1 at about 30 centimeters on the optical path. After opening the microscope shutter for the picosecond laser beam, adjust the mirror M1 to locate the picosecond laser beam center at the through hole of P1.Use an infrared scope to observe the position of the beam spot at P1 when adjusting the mirror M1.Similarly, adjust the mirror M2 to locate the picosecond laser beam center at the through hole of P2.Use the infrared scope to observe the position of the beam spot at P2 when adjusting the mirror M2.After adjusting the mirrors until the picosecond beam center locates at the through hole of both alignment plates, close the shutter of the picosecond beam in the microscope control software.

Next, open the microscope shutter for the femtosecond laser beam. Adjust the mirror M3 to locate the femtosecond laser beam spot center at the through hole of P1, then adjust mirror M4 to locate the femtosecond laser beam spot center at the through hole of P2.After adjusting the mirrors until the femtosecond laser beam center locates at the through holes of the two alignment plates, close the microscope shutter for the femtosecond beam. Next, place a camera at the position of the objective to visualize the location of the two beam spots and mark the position of the pump beam on the camera screen as a reference.

Then place an alignment plate P0 before the lens L3 and adjust the optical mirror one using a hex key so that the Stokes beam center passes the through hole of the alignment plate at the laser output port. Use the infrared scope to confirm the position of the beam spot at P0 during adjustment. Next, remove the alignment plate P0 and use the hex key to adjust the optical mirror two so that the center of the Stokes beam co-localizes with the reference mark of the pump beam on the camera.

Keep adjusting the mirrors until the Stokes beam strictly overlaps with the pump beam at both P0 and the camera planes. Open the lock-in amplifier control software and set the wavelength of the pump laser to 796 nanometers and the power of the pump and Stokes beam to 50 and 70 milliwatts corresponding to 15 and 25 milliwatts on the sample respectively. Then use olive oil for lock-in amplifier phase optimization.

The olive oil is sealed in a slide with tissue paper attached to the bottom to enhance the signal back scattering or EPI-SRS detection. Place the olive oil sample on the stage and adjust the focus of the 25X objective onto the sample. Using the microscope control software, set the imaging parameters as mentioned in the text manuscript.

Then using the lock-in amplifier control software, set the time constant value to 10 microseconds. Next, scan the laser beam over the sample, tuning the phase with a step-size of 22.5 degrees until the SRS signal intensity reaches the maximum. Then scan the sample with the laser shutter closed, tuning the offset value with a step-size of one millivolt until the average SRS signal is close to zero.

Next, open delay manager dialog in the OPO control software and scan the olive oil, tuning the delay stage until the olive oil SRS signal reaches its maximum. Then stop scanning and click on the add data button in the delay manager dialog to record the current delay data. After similarly acquiring delay data at different Raman shifts by imaging various chemical samples, select the data fit in order five on the delay manager dialog to fit the current data points with the fifth order polynomic function.

Then apply the fitted data by clicking on the use as custom button and checking the checkbox. The delay stage is auto-adjusted at different wavelengths according to the fitted delay curve. For in vivo imaging, fix the mouse on stabilization stage with its spinal cord exposed and immersed in saline.

Now mount the stabilization stage on a five-axis stage beneath the TPEF-SRS microscope. Then secure the mouse head with two head bars and lower the holding plate to offer enough space for chest movement during breathing, alleviating motion artifacts. Next, adjust the Z-translational stage to tune the focus until the Brightfield image of the spinal cord vasculature can be observed under a 10X objective.

Locate the spinal cord dorsal vein at the center of the field of view by tuning the two-axis translational stage of the five-axis stage. Next, tune the roll and pitch angles of the five-axis stage to adjust the spinal cord dorsal surface perpendicular to the objective axis based on the Brightfield image. Then replace the 10X with a 25X water immersion objective for TPEF-SRS imaging.

For SRS imaging, select the polarizing beam splitter above the objective by pressing the switch button connected to the motorized flipper. For TPEF imaging, select the dichroic mirror D2 above the objective by pressing the switch button connected to the motorized flipper. Finally, set the imaging parameters as described in the text manuscript and start scanning the sample.

In vivo dual-modal imaging of sparsely labeled YFP axons and myelin sheathes in the mouse spinal cord revealed that the axons are closely wrapped by a thick layer of myelin sheaths. As can be seen in the TPEF-SRS spinal cord image, the nodes of Ranvier have decreased axonal diameter and the axilemma is directly exposed to the extracellular matrix. Combined with new probes for fluorescence and SRS imaging two-photon excited fluorescence SRS microscopy can achieve simultaneous structural and functional imaging of various biomolecules, facilitating our understanding of complex biological processes.