Using Spectral Reflectometry to Determine Myelinated Axon Diameters in a Fixed Mouse Brain Slice

Take a hyperspectral confocal microscope with a stabilized laser source configured for a broad spectral range to decode the brain tissue myelin sheath nanostructure.

Use a reference mirror to capture the baseline reflectance spectrum. Remove the mirror and acquire a dark offset spectrum for detector noise correction.

Transfer a chamber with a fixed brain tissue section to the microscope stage.

Align the focal plane to the tissue region of interest, ensuring sufficient depth to minimize interference from the glass-tissue interface.

Laser light is directed onto the multilayered myelin sheath of axons.

As the light interacts with the myelin, partial reflections occur at the interfaces of the layers, producing interference patterns due to varying layer thicknesses and refractive indices.

Acquire spectral images of these interference patterns.

After baseline correction and signal analysis, the reflectance spectrum reveals the wavenumber periodicity, allowing the determination of myelinated axon diameters.