Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

We present a protocol for aligning in vivo vis-OCTF images with ex vivo immunostained retinal nerve fibers to validate our novel imaging technology. Vis-OCTF enables visualization of retinal nerve fibers in vivo, and this process confirms the accuracy of our findings. Our protocol combines in vivo and ex vivo imaging to provide a real-time visualization of retina structure and confirmed findings.

The process is applicable to most modals of eye diseases, provided the anterior eye portion allows for optical imaging. This includes diabetic retinopathy and retinal ischemia models. These studies lay the foundation for establishing an objective evaluation of neural damage in humans, which can significantly improve glaucoma diagnosis and treatment in the future.

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 axon bundle networks and surrounding retinal vasculature. Nucleate the eyes of a euthanized mouse, marking the temporal side for orientation purposes. Remove the anterior chamber, then remove the lens and vitreous, and place eye cups in paraldehyde for 30 minutes.

Wash eye cups with PBS for 30 minutes, replacing the solution three times. Then wash with 0.5%Triton X-100 for 30 minutes. Incubate eye cups in the blocking buffer for two hours at room temperature.

After immunostaining, isolate the retinas from the eye cups using a microscope. Divide the retinas into four leaves and flat-mount them with the retinal ganglion cell layer facing up. Ensure the mark on the temporal side remains attached for orientation.

Cover the retinas with mounting medium, place cover slips, and seal the slides using nail polish. Turn on the confocal microscope, and under Locate mode, find the area of interest using the eyepiece. Switch to Acquisition mode, and set up tiles to cover the entire retina along with Z-stack slices for all layers of information.

Image at least 25 tiles across the whole retina to achieve a total volume. Project the Z-stack slices to generate two-dimensional on-face confocal microscopy images. The composite vis-OCT fiber gram is compared with the corresponding confocal image of a 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 axon bundle networks and surrounding retinal vasculature. Start by creating a composite image using the four obtained images from each mouse.

Utilize a graphics editor to align all the blood vessels. Employ the optic nerve head and blood vessel patterns as landmarks for aligning the composite fiber gram. Overlap the composite OCT images and the confocal images of the same retina stained with ICAM II to achieve in vivo and ex vivo alignment.

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 axon bundle networks and surrounding retinal vasculature.