Preparing Retinal Organoid Samples for Transmission Electron Microscopy

1. Obtaining ROs from iPSCs²⁵

NOTE: ROs were derived from iPSCs by modifying the previously reported procedure.

1. Dissociate iPSCs at ~90% confluence using a bacterial protease (see Table of Materials). Chop up the colonies into pieces and scrape them with a cell lifter.
2. After collection, resuspend the clusters of cells in 0.25 mL of ice-cold Matrigel. Following 20 min of incubation at 37 °C, scatter the solidified gel with the DMEM/F12 medium (0.5% N2 supplement, 1% B27 supplement, 0.1 mM β-mercaptoethanol, 0.2 mM L-glutamine substitute, 100 U/mL penicillin and 100 mg/mL streptomycin). Define this time as day 0 of differentiation.
3. After 5 days of floating culture, cells group to form cell cysts. Transfer these cell cysts into a 100 mm Petri dish where they will now adhere and grow.
4. On day 15, use bacterial protease to dissociate the adhering cells and culture them in DMEM/F12 medium (2% B27 supplement, 0.1 mM non-essential amino acids).
5. On day 21, change the medium with 20 mL of serum-containing medium (DMEM/F12 medium, 8% FBS, 2% B27 supplement, 100 mM Taurine, and 2 mM L-glutamine substitute), and replace the medium every week.

2. Anterior fixation of ROs

1. Gently move one of the cultured ROs (~D180, a size of about 1~2 mm in diameter is optimal) from the Petri dish to 1.5 mL microcentrifuge tubes with a pipette and mark the relevant information about the ROs.
   NOTE: Cut ROs in half or cut off the poorly structured tissue with a razor under the microscope in the dish containing fixative if the ROs are too large.
2. Remove the culture medium from 1.5 mL microcentrifuge tubes with a pipette. Add 1.5 mL of 2% paraformaldehyde-2% glutaraldehyde fixative diluted in 0.1 M phosphate buffer (PB), pH 7.4, and fix at room temperature (RT) for 4 h. Store the microcentrifuge tubes at 4 °C overnight.
   NOTE: This step can be paused for 1 week but restarting as early as possible is recommended. If ROs are left for longer, it may cause autolysis of cells leading to destruction of the ultrastructure of ROs. Due to the fragility of the ROs, avoid rotating, oscillating, flipping, or harsh operations.

3. Post fixation of ROs

1. Remove the 2% paraformaldehyde-2% glutaraldehyde fixative and wash with 1 mL of 0.01 M phosphate buffered saline (PBS) at pH 7.4 for 3 x 10 min. Gently suspend the ROs in the 1.5 mL microcentrifuge tubes during the washing process for fully rinsing. For every additional day of fixation, increase the number of washes by two to remove the residual fixative as much as possible.
2. Remove any remaining PBS using a pipette and replace it with ~150 µL of 1% osmic acid (OsO4) until the tissue blocks are submerged. Place the microcentrifuge tubes in a dark box to protect them from light and infiltrate at RT for 1 h.
   NOTE: OsO4 is a strong oxidant and must be handled in a fume hood.

4. Staining and dehydration

NOTE: The dehumidifier must be turned on to dry the environment from this step.

1. Remove OsO4 using a pipette, wash with 0.01 M PBS (pH 7.4) 3 x 10 min, and then wash 3 x 10 min with deionized water.
   NOTE: To ensure complete rinsing of ROs, we recommend suspending the ROs. Aspirate OsO4 into a dedicated disposal bottle.
2. Remove the last deionized water wash and replace with ~150 µL per tube (ensure full infiltration of tissue blocks) of uranium acetate and stain for 1-2 h at RT. Protect the microcentrifuge tubes from light.
3. Remove the uranium acetate and replace it with 1 mL of 50% acetone in the fume hood, dehydrating for 10 min. Then, use 70%, 80%, 90%, 100%, and 100% acetone for gradient dehydration of the ROs successively. Each dehydration lasts 10 min.
   NOTE: Leave some liquid to soak the ROs for each exchange to prevent exposure to air and moisture. Additionally, operate quickly as soon as possible because acetone is volatile. Aspirate uranium acetate into a dedicated disposal bottle.

5. Infiltration

1. Discard the acetone and add ~150 µL of the mixture consisting of acetone and Epon-812 resin at a ratio of 1:1. Place it in a 37 °C oven for 1 h.
2. Remove the above mixture and replace it with a mixture consisting of acetone and Epon-812 resin at a ratio of 1:4. Place it in a 37 °C oven overnight.
3. Transfer the ROs into the new tubes containing ~500 µL of pure epoxy resin carefully and place them in a 45 °C oven for 1 h.

6. Embedding

1. Prepare the 30-50 mL of pure epoxy resin in a 50 mL centrifuge tube by mixing the reagent kit according to the manufacturer's instructions in advance. Rotate the pure epoxy resin upside down slowly for 30 min to reduce air bubbles introduced by the mixer. Then, leave it in a 37 °C oven for 20 min. Add 2/3 volume of pure epoxy resin to the groove of the embedding mold.
2. Use a toothpick to pick ROs into the embedding mold equipped with pure epoxy resin.
3. Fill the groove with pure epoxy resin until it slightly protrudes or bulges.
4. Adjust the position of ROs at both ends of the embedding mold for directional embedding. Place the embedding mold in a 45 °C oven for 1-2 h and then put it in a 65 °C oven for 48-72 h.

7. Semi-thin positioning

1. Sand the excess epoxy resin around the tissue from the ends of the longer four sides of the embedding block to form a trapezoidal face. 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.
2. Trim away the excess resin until the desired area is exposed.
3. Fill the trough of the glass knife with water. Cut semi-thin slices to a thickness of 1 µm using a semi-thin microtome and lay them on a microscope slide with a needle.
4. Add 50 µL of 1% toluidine blue to dye semi-thin slices and incubate for 1 min at 95 °C on the heating plate. Rinse with distilled water for 2 min. Use a 20x ordinary optical microscope to observe the structure of the sections.

8. Ultra-thin sectioning

1. Install the embedding blocks and glass knives as described in step 7.1. Adjust the distance between the knife and the tissue block, the liquid level of the sink, and the slicing speed. Cut the ultrathin sections with a thickness of ~70-100 nm with an ultrathin microtome and scoop the slices onto the copper mesh (200 mesh) with the supporting film.
2. Stain ultrathin sections with 3% lead citrate for 15 min to enhance contrast in a dual staining procedure.

9. Imaging ROs by TEM

1. Launch the software (see the Table of Materials).
2. Click on the Memory mode button and mark each ultrathin section roughly.
3. Search for synaptic structures.
   1. Distinguish the laminated structure of ROs at low magnification.
   2. Locate the outer nuclear layer (ONL), which shows a nuclear structure with a deeper electron density. Subsequently, navigate to the terminal of the photoreceptor where numerous vesicles accumulate. Search for the rod terminals with a single synaptic ribbon and the cone terminals with multiple synaptic ribbons.
   3. Move to the inner plexiform layer (IPL) and seek out a similar structure with a small ribbon.
4. Capture the microscopic image.
   1. Click Start and input the corresponding magnifying power.
   2. Save the images.