C1 system

The eyeballs were cut in half at the ora serrata and the anterior segments were immersion fixed in 4% paraformaldehyde in 0.1M PB for 30 minutes at room temperature, followed by buffer immersion wash for another 30 minutes. Then the perfused half of iris was carefully dissected out from the root connected to the ciliary body, and cut into 4 to 5 sectors. The posterior pigment epithelial layer of iris was carefully removed with the aid of dissecting microscope. The iris sectors were immersed in RapiClear® 1.47 (Sunjin Lab, Taiwan) for 2 hours for optical clearing before it is flat-mounted with anterior face up for confocal imaging.
Confocal imaging was performed on Nikon i90 microscope and Nikon C1 system with Nikon) , equipped with 4 (408 nm, 488 nm, 546 nm and 635 nm) laser board. Confocal images of all iris sectors were acquired at low magnification first (x2 and x4, Nikon Plan Apo lenses), to help orientate and identify the areas of interest. Specific regions were zoomed in with high-power objective lens (x40, Nikon plan apochromatic oil lens, NA1.0) for detailed imaging of labelled structures at cellular level. Z-stacks were captured at 0.3 µm optical section increments along the z-plane for each order of arteries and veins, according to our classification with the aid of silver staining published previously.

After perfusion labeling, the eye was left immerse fixed overnight in 4% paraformaldehyde (in 0.1M phosphate buffer). The retina was dissected out the next day and flat-mounted onto glass slides using glycerol. The correct orientation of the retina for imaging was determined via the naming convention of the eye bank for left and right eyes, by visual confirmation of external landmarks from remnants of ocular muscle insertion points, 33 the inferior location of the CRA as well the retinal vascular distribution pattern upon flat mounting.
Confocal images were collected from the macula region on the Nikon C1 system using 310 Plan Apo objective lens. As far as possible, image stacks were collected at 1.15-lm step size in the four ''quadrants'' of the macula; namely, superior, inferior, temporal, and nasal (direction toward the optic disk) in relation to the foveola (Fig. 1). Confocal images were collected at 1024 3 1024 pixel resolution for each image field measuring 1270 3 1270 lm (1 pixel ¼ 1.24 lm). Each image stack is collected to include the uppermost (vitread) and outermost (sclerad) retinal vessel that can be detected.