Our research primarily focuses on analyze the synaptic autostructure in mature retinal organoids. We aim to develop a reproducible and a straightforward transmission electro microscopy, or TEM sample preparation method, for retinal organoids, which could greatly benefit the field of retinal organoid research. Compared to our techniques, such as optical microscope, transmission electron microscope offers the ability to characterize the autostructure of integral synapse and the subcellular analysis in retinal organoids at the nanoscale level.
Our protocol offers of optimize the conditions for transmission electron microscope sample preparation of retinal organoids, which are in simple and user-friendly steps. Our findings clearly show the ultra structure morphology of synapse within retinal organoids, including both conventional synapse and ribbon synapse. This establishes our solid foundations, and presents a reproducible methods for subsequent functional investigations of retinal organoids.
Along with the evaluation of the clinical applications, such as jump screening. To begin the anterior fixation of the induced pluripotent stem cell derived retinal organoids or ROs, using a pipette, gently move one of the cultured ROs from the Petri dish to a 1.5 milliliter micro centrifuge tube. With the help of a pipette, remove the culture medium from the micro centrifuge tube and add 1.5 milliliters of 2%paraformaldehyde, 2%glutaraldehyde fixative.
After the ROs are fixed, remove the paraformaldehyde glutaraldehyde fixative before proceeding to wash the organoids. Gently suspend the organoids in the microcentrifuge tube by adding 1 milliliter of 0.01 molar PBS to ensure complete washing. Then, pipette out any remaining PBS and replace it with approximately 150 microliters of 1%osmic acid until the tissue blocks are submerged.
Place the microcentrifuge tube in a dark box. To begin staining the fixed retinal organoids or ROs for transmission electron microscopy, use a pipette to remove the osmic acid previously added to the organoids in the micro centrifuge tube during post fixation. Then, wash the ROs with 0.01 molar PBS at pH 7.4 three times for 10 minutes each, followed by washing with deionized water three times for 10 minutes each.
Remove the last deionized water wash. Replace it with about 150 microliters of uranium acetate and stain for one to two hours at room temperature. Next, in a fume hood, remove the uranium acetate and replace it with one milliliter of 50%acetone to dehydrate the samples for 10 minutes.
Then, perform gradient dehydration using 70%80%90%and 2 times 100%acetone successively, each for 10 minutes. After the gradient dehydration, discard the residual acetone and add approximately 150 microliters of a mixture of acetone and Epon-812 resin. Place the tube in a 37 degree Celsius oven for one hour.
After removing the previous acetone resin mixture, replace it with a different composition of acetone and Epon-812 resin mixture. This time incubate the tube at 37 degrees Celsius overnight. Finally, transfer the retinal organoids into a new tube containing approximately 500 microliters of pure epoxy resin, carefully.
And incubate it at 45 degrees Celsius for one hour before proceeding with organoid embedding. To begin, add pure epoxy resin to the groove of the embedding mold up to two thirds of its volume. Use a toothpick to pick retinal organoids and transfer them into the embedding mold equipped with pure epoxy resin.
Then, fill the groove with pure epoxy resin until it slightly protrudes or bulges. Adjust the position of the organoids at both ends of the embedding mold for directional embedding. Install the embedding block into the sample mounting stand and screw down the bolt with an L screwdriver.
Then install glass knives on the knife stand and tighten the nut manually. Trim away the excess resin until the desired area is exposed. Fill the trough of the glass knife with water.
Using a semi thin microtome, cut semi thin slices to a thickness of one micron and lay them on a microscope slide with a needle. Add 50 microliters of 1%toluidine blue to dye the semi thin slices, and incubate for one minute at 95 degrees Celsius on the heating plate. Rinse with distill water for two minutes.
Use a 20x ordinary optical microscope to observe the structure of the sections.
