1. Preparing Dishes with EM Grids for Plating Primary Neurons

1. Examine the integrity of holey carbon on the gold EM grids using a light microscope at magnification of at least 25X. Make sure carbon holes are >98% intact.
2. For plating primary neurons, use a Bunsen burner to flame-sterilize the EM grids and concurrently render them hydrophilic. Use tweezers to pick up the EM grid by its edge, not the central gridded area. CAUTION: Never leave a lit Bunsen burner unattended, and do not use gloves or tweezers with plastic components while performing these steps:
   1. Before lighting the Bunsen burner, set the air and gas adjustments to a minimally open position.
   2. Light the Bunsen burner using a striker flint or butane lighter.
   3. Modify the air and gas adjustments to achieve a small, blue inner flame within a taller, lighter blue/violet flame. The tip of the inner flame is the hottest part of the flame. The knob underneath the burner adjusts the amount of gas entering the burner tube, while the barrel of the burner can be turned to adjust the amount of air entering the burner. Turning the air adjustment clockwise decreases the air (resulting in a purple flame) and counterclockwise increases the air (resulting in a yellow flame).
   4. Using metal tweezers (no plastic parts) to hold the EM grid, pass it quickly through the flame twice, facing carbon-side up. The carbon side will appear more matte (less shiny) and with more of a grayish tint than the other side.
   5. Immediately transfer the grid (carbon-side up) into the center of the glass-bottom dish placed within 10 cm of the Bunsen burner. Use one EM grid per dish (Figure 1). Only use glass-bottom dishes that are presterilized, i.e. via gamma irradiation.
3. Use a light microscope to check the grid integrity (carbon holes intact) whilst still keeping the EM grid inside the glass-bottom dish (to avoid contamination). When applying any substance on the grid or anything that will come in contact with the neurons, use sterile procedure and sterile pipette tips.
4. In a tissue culture hood using sterile procedure, apply 250 µl of the appropriate coating substance slowly and carefully to the central glass area of the Petri dish. Make sure the appropriate coating substance covers the entire EM grid.
   1. For hippocampal neurons, use poly-L-lysine (PLL, 1 mg/ml) as the coating substance, prepared as previously described¹⁹. Note that 250 µl are needed per EM grid, per dish, so scale the batch accordingly. For dorsal root ganglion (DRG) neurons, use a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (see Table of Materials and Reagents) as the coating substance, prepare as follows. Note that 250 µl are needed per EM grid, per dish, so scale the batch accordingly.
      1. Thaw a stock bottle of the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells overnight at 4 °C.
      2. Keep cold the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, and all components used to make the solution, i.e. by keeping them on ice.
      3. While on ice, dilute this gelatinous protein mixture in Neurobasal medium to yield a 1:20 dilution (i.e. dilute 500 µl of the gelatinous protein mixture into 10 ml of Neurobasal).
      4. Divide diluted gelatinous protein mixture into 1 ml aliquots, and immediately store any extra aliquots at -20 °C.
      5. Apply 250 µl per EM grid, per dish, as described in Section 1.4.
5. Cover the Petri dish with its top and incubate (either with PLL for hippocampal specimens, OR with the gelatinous protein mixture for DRG specimens) for 1 hr at room temperature in the tissue culture hood.
6. Aspirate all PLL (for hippocampal specimens) or the gelatinous protein mixture (for DRG specimens) from the dishes. To do this, use the vacuum system in the tissue culture hood. Use a sterile pipette tip attached to the vacuum tube. Avoid direct contact with the EM grid.
7. Use an adjustable air-displacement pipette to carefully apply 250 µl of sterile PBS (phosphate-buffered saline) to the EM grid in the central glass area of each Petri dish, such that the EM grid is fully covered by PBS. Then, aspirate the PBS from each dish. Repeat 3x.
8. Allow the dishes with EM grids to dry under the tissue culture hood for 15 min. Make sure they are completely dry by checking under the light microscope in the tissue culture room. Make sure there are no bubbles of moisture in the grid. If so, carefully aspirate next to the EM grid to eliminate this extra moisture. The coated grid should be used immediately for plating the neurons.

2. Preparing and Plating Primary Neurons on EM Grids

1. To plate primary neurons, first dissect, trypsinize (using 2.5% trypsin) and triturate to dissociate the hippocampi (or dorsal root ganglion of 18-day old rat embryos) into individual cells, as previously described¹⁹.
   1. Triturate is a common term used by neurobiologists to describe dissociating clumps of cells into individual cells, particularly by use of a glass pipette with a fire-polished tip. The result is achieved by carefully and slowly drawing and releasing the cells, up and down, multiple times (~30) through the pipette.
2. Follow procedures as previously described for DRG dissection and cell isolation²⁴, taking note to use 0.25% trypsin (in HBSS) to isolate the rat DRG neurons, rather than use the enzymes suggested (papain, collagenase/dispase) that are more suitable for mouse DRG neurons.
3. Calculate the number of isolated neurons (i.e. using a hemocytometer) and use this value to calculate the appropriate volume of cells to apply to each dish to achieve a concentration of 50,000 cells/ml per dish. The maximum volume to apply is 250 µl. Note that this only fills the central glass area, not the entire dish.
4. Incubate the dishes for 30 min in a CO₂ incubator at 37 °C. Allow cells to recover and adhere.
5. Slowly add 1.5 ml of warmed media to each dish, taking care not to disturb the EM grid. The type of media depends on the cell type (hippocampal or DRG).
   1. Prepare the appropriate media for either hippocampal neurons¹⁹ or dorsal root ganglion neurons²⁴, which is to be warmed in a 37 °C water bath prior to use. Incubate the dishes overnight in the CO₂ incubator.
   2. For hippocampal neurons, change the media the next day. Change half of the media every two days onward for 14 days. Warm the media in a 37 °C water bath prior to use.
   3. For DRG neurons, the day following dissection/plating, prepare a fresh stock of media with an anti-mitotic agent (Uridine and 5'-Fluoro-2'-deoxyuridine) make a 10 mM stock solution of each, separately; use a final concentration of 10 µM for each. Remove half of the DRG media (875 µl) and add 875 µl of fresh media with the anti-mitotic agent.
      1. For DRG neurons, every two days for the first week, alternate changing media between anti-mitotic media and standard DRG media. For the second week, change standard DRG media every two days.

3. Vitrifying Neurons on EM Grids

1. Prepare equipment and all materials for freezing and storing the gold EM grids at cryogenic temperature: a vitrification device with a humidity chamber¹⁷, fine-point specialized tweezers for the vitrification machine, long flat point tweezers, dewar(s) of liquid nitrogen (LN₂), coolant container, EM grid storage box, calcium-free filter paper.
2. Start the vitrification device. Set the humidity to 100% and temperature to 32 °C. In the “Console” section, set the blot time to zero seconds. This allows for manual blotting through the side-window of the vitrification machine's humidity chamber.
3. Handle the calcium-free filter paper with gloves, layering them such that three papers are stacked. Cut the stack into 0.5 cm wide strips that are ~2 cm long. Bend them at a 90° angle such that one side of the paper has a 0.5 cm x 0.5 cm face (Figure 2). Using tweezers, remove the middle paper and place it on another calcium-free filter paper until use.
4. Put the grid storage button in the button holder within the vitrification chamber. Fill the inner chamber of the coolant container with liquid nitrogen and wait until complete evaporation. Fill the outer chamber of the coolant container with liquid nitrogen until it attains stable temperature for proceeding to the next step. Fill the inner chamber with high-purity gaseous ethane that will condense to a liquid state within the cooled chamber.
5. Move the dish(es) from the incubator to a large 100 mm polystyrene dish for transport to the vitrification room, if not within the immediate vicinity. Use the specialized vitrification tweezers to carefully pick the EM grid from the dish. Note which side the neurons are growing on the EM grid; the position will matter for the next step. Use the black sliding lock on the tweezers to securely lock the tweezers on the EM grid.
6. Insert the tweezers into the vitrification machine such that side of the EM grid on which the neurons are adhered faces to the left, away from the side-opening hole of the vitrification machine. Retract the specialized tweezers into the vitrification machine.
7. Place the coolant container in the appropriate holder of the vitrification machine. It should be filled with adequate LN₂ and liquid ethane. Using the appropriate screen command of the vitrification machine, raise the coolant chamber upward until it's flushed with the bottom of the humidity chamber.
8. With the flat point tweezers, grasp one edge of the filter paper such that the shorter side (the 0.5 cm x 0.5 cm face) is perpendicular to the tweezers. This face will come into direct contact with the EM grid for blotting (Figure 2B). Carefully insert the filter paper into the side-hole of the vitrification machine's humidity chamber (Figure 2A). Stably hold the paper against the EM grid (the side facing away from the specimen) for 10 sec. Discard the paper afterward and immediately plunge-freeze the specimen in the liquid ethane using the vitrification machine's automation.
9. Carefully transfer your frozen-hydrated EM grid to one of the slots in one of the grid storage buttons. Repeat the process for additional EM grids in the dishes. The EM grid storage buttons used in these experiments can store multiple frozen-hydrated EM grids.

4. Image Collection, Processing, and Annotation

1. Proceed to collect 2-D electron micrographs and/or 3-D tilt series⁵ of the neurites using a cryo-electron microscope under a low-dose condition. The 2-D images were intended to assess the quality of the grid in terms of ice thickness and the possible areas to be useful for 3-D tilt series.
   1. In this case, all images were collected using a 4k x 4k CCD camera attached to a 200 kV electron microscope equipped with a single tilt liquid nitrogen cryo transfer holder, at 20k microscope magnification at a target underfocus of 7 µm and sampling of 4.4 Å/pixel.
   2. To obtain a 3D tomogram of the sample, take a series of projection images while incrementally tilting the sample along one axis of the transmission electron microscope (TEM). Given the tilt angles and other experimental settings, there are several different softwares available to automatically collect the tilt series⁵. The 3-D tilt series shown here were collected on the same microscope using semi-automated tilt series acquisition software 26 over a range of -60° to 60° at 5° increments with a cumulative dose of ~60 e/Ų.
2. Reconstruct the tilt series of the neurites using image processing software²¹ as previously described for other samples^5,29.
3. Color-annotate the 3-D features of the neurites by first segmenting the tomogram and creating a surface model using a 3-D image processing software as previously described⁵.