Preparation of Retinal Samples for Volume Electron Microscopy

We focus on examining the nanoscale 3D structural features of photoreceptor terminals in the mouse retina. Our primary goal is to design a 3DEM sample preparation method, suitable for a small volume of biological samples, and we have reconstructed the terminal structure of retinal photoreceptors using FIB-SEM. Using the retinal 3DEM samples with the OTO method, we segmented and reconstructed the 3D structure of four types of photoreceptor terminals, and subsequently analyzed the relationship between their components.

Our retinal sample preparation protocol is user-friendly, requires no extra equipment and reduces serious processing time. However, there may be some blurriness in the details of the cell membranes, which we will try to adjust. In the future, we hope to further explore the function on each component within the retinal terminal and its role in synaptic activity in To begin, place the euthanized mouse on the operating table.

Using curved scissors, excise the eyes under dim red light and immediately place them in the fixation solution. After removing the anterior segment and vitreous from the eye, isolate the retina completely. Under a dissection microscope, quickly slice the retina into 100 to 200 micrometer thick strips and transfer them into a new microcentrifuge tube containing fixation solution.

Incubate the tube in a rotator for two hours at room temperature, and then place it at four degrees Celsius for 24 hours. To begin, obtain the retinal strips for mice and wash them five times in 0.1 molar sodium cacodylate buffer for 15 minutes. Then, incubate the strips with potassium ferrocyanide and osmium tetroxide solution in the dark on ice for 1.5 hours.

Wash the strips three times in double distilled water and incubate them with 1%thio-carbo-hydrazide. After rinsing with double distilled water, sequentially treat the retina strips with osmium tetroxide, uranyl acetate, and lead nitrate. Dehydrate the retina strips with increasing concentrations of ethanol.

Then, treat retina strips with a solution containing equal ethanol and acetone for 20 minutes, followed by two acetone washes for 20 minutes each. Sequentially, incubate the retina strips in acetone resin mixtures in the dark at room temperature. Infiltrate the retina strips with pure resin for 24 hours at 45 degrees Celsius, followed by fresh resin for 48 hours at 60 degrees Celsius.

The focused ion beam scanning electron microscopy of retinal photoreceptor terminals, using this method preserves cell membrane structure and vesicle outlines more clearly than the traditional double fixation method.