Coherent Anti-Stokes Raman Spectroscopy to Visualize Myelinated Neurons in Mouse Brain Tissue

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