Preparing Porcine Eyes for Confocal Reflectance Microscopy to Visualize the Vitreous Collagen Fiber Network

Changes in the vitreous lead to retinal detachment. We think that changes in the vitreous collagen fiber network change gel stiffness, causing focal traction on the retina. It's hard to image vitreous.

Traditional histology causes shrinkage, loss and a honeycomb artifact since the vitreous is more than 98%water. Most tissues are under 80%water. Our imaging strategy preserves the natural arrangement of collagen fibers in the vitreous since it requires minimal processing, only gross dissection.

We hope that this strategy will allow us to discover the root cause of retinal detachment and provide more effective ways to prevent it. To begin, obtain ex vivo porcine eyes from a meat processing plant. Using dissecting scissors or forceps, remove any excess muscle and connective tissue from the globe.

Now, position a three-pronged pedestal at the base of a cube mold, such as an extra large silicone ice cube mold, and position the globe on the pedestal, ensuring the limbus rests securely on the prongs. Next, prepare a 3%agarose gel by adding three grams of general purpose agarose to 100 milliliters of distilled water and heat the mixture until fully dissolved. Allow the solution to cool to approximately 37 degrees Celsius and pour it into the mold until the globe is completely submerged.

Place the agarose cube in a refrigerator and chill for at least four hours. After chilling, remove the solidified agarose cube from the mold and carefully remove the pedestal from the base of the cube. Construct a three-sided frame matching the dimensions of the agarose cube using one-by-eight LEGO bricks, plates, and tiles.

Then place the chilled agarose cube in the LEGO frame. Hold a high-profile microtome blade by the blunt edges and align it with the top of the LEGO frame. Apply gentle downward pressure and drag the blade back and forth through the entire cube and embedded globe.

Using a microtome blade, trim approximately one millimeter of agarose from the entire cut surface of the cube. Use a tissue wipe or a small triangular cellulose sponge to gently dry the cut surface of the agarose cube, focusing near the border of the eye cap. Now, add approximately one teaspoon of all-purpose silicone caulk to a small plastic sandwich bag and cut a three millimeter hole in one of the bottom corners of the bag to create a makeshift piping bag.

Using the piping bag, apply a thin bead of silicone caulk along the cut surface of the agarose cube, precisely around the edge of the eye cap, to form a waterproof seal. Ensure that the caulk forms a complete and continuous border to prevent vitreous gel leakage. Then place a large cover slip glass over the eye cap and press it down until the surface of the eye cap makes full contact with the glass.

Confirm that the silicone caulk forms an unbroken seal with the cover slip. Next, while holding the agarose cube, invert the sample and place it on a large cover slip adapter suitable for imaging on an inverted microscope. Use an inverted confocal microscope that is not a spinning disc type.

Select an oil objective for imaging. Adjust the light path settings on the confocal microscope to obtain reflectance images. Replace the dichroic mirror with a beam splitting mirror that redirects part of the illuminating laser and transmits a portion of the reflected light.

Then set the detection wavelengths to include the wavelength of the illuminating laser. To orient the Z position within the sample, reduce the laser power to its lowest possible setting. Identify the brief flash of reflected light that appears at both the top and bottom of the cover slip to mark the Z position boundaries.

Increase the laser power to a level higher than typically used for fluorescence imaging. Finally, in the image acquisition settings, activate line averaging and increase the pixel dwell time to improve the signal to noise ratio and acquire the images. One eye each from three young adult pigs was imaged after sectioning the eye in an axial plane through the inferior limbus.

Representative confocal reflectance images showed visually denser fiber networks in pigs 1 and 2 compared to pig 3. The total length of vitreous fibers per tile was similar across all three pigs, indicating no significant difference in fiber abundance, but the fraction of pixels occupied by signal differed between the three pigs. The curvature of the fibers, which is the mean incremental change in angle along a fiber, and the lacunarity were similar between the three pigs.

The hyphal growth unit defined as branch points per fiber length was highest in pig 3, differing significantly from pigs 1 and 2.