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.

Mount a reference mirror on the microscope stage, with the surface facing the objective lens. If it is not easy to put the mirror on the microscope stage, adhere a mirror onto the flat plate. Adjust the microscope stage to align the focal plane to the mirror surface. Then, adjust the PMT gain and the laser power, considering the dynamic range of the detector.

Under a pseudocolor, check that there is no saturation throughout the wavelength range. If saturation is observed, lower the laser power. Then, run the lambda scan acquisition. Remove the mirror from the stage, and repeat the same acquisition without a sample in order to obtain the dark reference. Then, save the data in a multi-stacked TIFF format.

To carry out SpeRe image acquisition, place the mounted tissue on the microscope stage. To roughly align the tissue to the focal plane of the objective lens, through an eyepiece, use wide-field fluorescence mode. With the live scan on, control the microscope stage to align the focal plane to the region of interest in the tissue. To avoid background noise from the coverslip, select a target region of at least 15 micrometers in depth from the glass tissue interface.

Acquire the spectral image stack for the target region using the same procedure as demonstrated earlier in this video. Then, save the data for the tissue and the dark offset in multi-stacked TIFF format. To process the images in ImageJ, open the spectral data for the reference mirror and brain tissue.

Select the ROIs for the opened image stacks, the central area for the reference mirror, and the segment of an axon fiber for the brain tissue. Then, run image, stacks, plots z-axis profile to acquire the raw spectra for the selected ROIs.

The axon fibers may be structurally heterogeneous along their lengths, hence selecting the ROI on a small axon segment, typically less than 5 micrometers, is recommended to minimize partial volume artifacts.

Open the dark offset data, one taken for the reference mirror, and the other taken for the brain tissue, and plot the z-axis profile as just demonstrated. Then, use the copy and paste options to save all acquired options.