This technique can help answer key questions in the myelin field, such as myelin plasticity and demyelination. The main advantage of this technique is that it allows the study of the nanostructure of myelinated axon in intact brain tissue without any complex labeling or a sample preparation. Here, we will demonstrate on the application in a brain slice, but this technique can be extended to living animals.

After fixing and slicing mouse brain tissue according to the text protocol, prepare one glass slide and two cover glasses for each tissue slice. Use a glass cutter to cut one of the square cover glasses in half. Then, create a spacer by using superglue to attach the two pieces onto the glass slide.

Using a brush or a pipette, harvest a brain slice and lay the tissue on a glass slide between the spacer, taking care not to fold the tissue. Dispense 100 microliters of PBS on the tissue surface. Then, place the second square cover glass on the top of the tissue while avoiding the introduction of air bubbles.

Use nail polish to seal around the cover glass to prevent evaporation of PBS and contamination by dust during the subsequent imaging session. At least one hour before imaging, switch on the microscope to allow thermal stabilization of the laser source. Then turn on the software for spectral scanning and click the acquire button on top of the GUI.

Turn on the software shutter for the white light laser and photomultiplier tube. Under acquisition mode, select the XY lambda mode on the dropdown list. The laser input mode is then automatically changed from constant percent to constant power.

Under lambda excitation, lambda scan settings, uncheck the automatic SP movement box and set the spectral window for the input laser to 470 to 670 nanometers and the spectral step size to four nanometers. Set the spectral range of PMT to 450 to 690 nanometers by double-clicking or moving the adjustment bar for spectral range. Select a water-immersion objective lens suitably with a high numerical aperture.

Give any value to the PMT and laser power to activate the AOBS configuration and live scan button. Under AOBS configuration, switch to the optical path by checking reflection. Next, mount a reference mirror on the microscope's 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's stage to align the focal plane to the mirror's 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 TIF format. To carry out SpeRe image acquisition, place the mounted tissue on the microscope's 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's 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 TIF 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 five 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. To carry out baseline correction and SpeRe signal analysis, subtract the offset spectra from the spectra of the reference mirror and the brain tissues.

Normalize each spectrum by dividing the maximum intensity of the spectrum. Enter the wavenumber by taking the reciprocal of wavelength used in experiment, normalize the spectrum of the axon by dividing that of the reference mirror, and subtract DC offset from the normalized spectrum. And then obtain the wavenumber frequency by fitting the acquired spectrum to a sinusoid.

Convert the acquired wavenumber frequency to periodicity by taking the reciprocal and convert the wavenumber periodicity to the axon diameter using the equation shown here. In this experiment, fixed brain slices were fluorescently stained against myelin and SpeRe imaging was carried out with a light dose an order of magnitude lower than conventional fluorescence confocal microscopy. The SpeRe signal was localized along the center of the myelinated axons as expected by the geometry of the optical reflection.

From the reflectant spectrum of an axon segment, the wavenumber periodicity was obtained, which was subsequently converted to axon diameter. This graph illustrates a transversal profile of fluorescence intensity from the boxed region here, and the diameter measured by SpeRe was found to be in good agreement with the fluorescence-based measurement. The SpeRe imaging is based on optical reflection.

Thus, a silica-based coverslip can introduce significant background noise. In the optics setup, the background noise was considerable when the imaging depth from the coverslip was less than five micrometers but was avoided when the imaging depth is greater than 15 micrometers. The calibration step is not required for every experiment but is recommended at least once a week.

Once calibration is completed, the imaging procedure can be finished typically within 20 seconds for each field of view. While attempting this procedure, it is important to remember to consider the dynamic latency of the detector for full-spectra scanning. After watching this video, you should have a good understanding of how to obtain the nanostructure from myelinated axons using reflectance images.