This article describes a method for imaging neuronal architecture in mouse brain slices using confocal microscopy and coherent anti-Stokes Raman spectroscopy (CARS). The technique allows for the visualization of myelin sheaths and Nissl bodies, providing insights into neuronal structure.
Take a chemically fixed mouse brain slice in a multiwell plate.
The tissue contains axons surrounded by lipid-rich myelin sheaths. The cell bodies contain Nissl bodies composed of rough endoplasmic reticulum and ribosomes.
Incubate in a permeabilization solution containing a fluorescent Nissl stain. The solution permeabilizes cellular membranes, allowing the stain to bind to the ribosomal RNA within the Nissl bodies and label the cell bodies.
Place the tissue under a confocal microscope with a coherent anti-Stokes Raman spectroscopy, or CARS, imaging system.
Direct synchronized pump and Stokes laser beams onto the tissue, tuned to excite molecular bonds in the lipid-rich myelin sheath.
Introduce a probe laser beam to interact with the excited bonds and produce an anti-Stokes signal specific to myelin.
A photodetector captures the signal to generate a myelin-specific image.
Use confocal imaging to capture signals from the labeled Nissl bodies.
Overlay the confocal and CARS images for visualizing neuronal architecture.
After preparing the tissues as described in the manuscript, stain free-floating sections for Nissl and antibody media to visualize cell bodies. Then, incubate the sections on a standard laboratory shaker for 30 minutes at room temperature. Before bringing the samples to the microscope, turn on and warm up the CARS laser for at least one hour. Then, align the CARS laser by spatially overlapping the pump and Stokes laser beams, and adjust the delay between the two laser beams.
Kohler the condenser optics and the diaphragm of the microscope for forward CARS imaging. Then, adjust the external periscope to center the spatially overlapped two lasers onto the scanning head mirrors of the microscope. For best forward CARS non-descanned detection, make sure the condenser is kohlered.
For immunofluorescence confocal imaging, and CARS imaging, fit the CARS laser with both forward and epi-CARS non-descanned detectors by incorporating a confocal microscope equipped with visible lasers for fluorescence imaging. Now, place the sections in a culture dish with a coverslip bottom and PBS to avoid drying the tissue. Also, use a glass weight to keep the tissue near the coverslip.
Using the graphic user interface. Set the image acquisition parameters for CARS and Nissl fluorescence confocal imaging. Place the sample on the microscope stage. Focus the sample and capture the images.
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