Start by positioning the anesthetized mouse on the animal holder and securing it with two Velcro straps. Turn on the computer and open the software to activate the laser. Adjust the animal holder to center the laser in the mouse's eye.
Visualize the posterior part through an on-face preview, the field of view of the superficial vascular plexus, a B scan, and the retina cross-section within the field of view. Acquire a visible light optical coherence tomography, or vis-OCT volume after making minor adjustments to the optical focus. Align the optical nerve head in each of the four corners of the field of view to cover different retinal areas.
Use an intensity-based threshold method to detect the retina surface, and generate vis-OCT fiber grams from the volume. Crop the retinal nerve fiber layer, or RNFL, by selecting the first 16 micrometers depth. Next, calculate the mean intensity projection along the axial direction to generate the fiber gram image composed of retinal ganglion cell axon bundles and surrounding vasculature.
Align the blood vessels using a graphics editor to montage the four images post-processing. The composite vis-OCT fiber gram is compared with the corresponding confocal image of flat-mounted retina immunostained with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM II labeled blood vessels on the confocal image.
Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC exon bundle networks and surrounding retinal vasculature.
