The overall goal of this procedure is to capture membrane dynamics by electron microscopy following induction of neuronal activity using optogenetics then freezing cells at defined time points after stimulation. This method can help answer key questions in neuroscience and cell biology about the mechanisms of synaptic vesicle recycling and also maybe modeling on a very rapid time scale. The main advantage of this technique is being able to visualize morphological changes within a cell using electron microscopy on a millisecond time scale.
The procedure will be demonstrated by Shuo Li, a graduate student and Sumana Raychaudhuri, a research associate. Both from my laboratory. Before preparing the fixative, label two milliliter cryogenic files with a pencil according to their specimen numbers.
Then combine all of the fixative reagents, and aliquot one milliliter of volumes of the fixative into each vial. After capping the tubes, immediately submerge fixatives in liquid nitrogen and store the vials in a closed styrofoam box. Next, fill an automated free substitution unit and high pressure freezer with liquid nitrogen.
Then place a small container of acetone into the specimen chamber of the free substitution unit and wait until the temperature of the substitution unit reaches minus 90 degrees Celsius. To program the light stimulation protocol, click edit in the light stimulation window of the touchscreen monitor. Another window will open.
Enter 15 seconds for the dark phase. 100 milliseconds for the period. 10 milliseconds for the pulse, and one for the number of periods for a single 10 millisecond stimulus.
On the main screen, confirm that the light stimulation box is checked, and click specimen storage followed by edit. In the new window, use the plus or minus button to select two to store two specimens in each channel and confirm that storage liquid nitrogen enabled is elected. Now under a stereo microscope, place a sapphire disc cell side up in the well of a middle black plate.
Followed by a 100 micron spacer ring. Dip one side of a blank sapphire disc into pre-warmed saline solution and place the disc over the spacer ring, saline side down. Place another 100 micron spacer ring.
And a 400 micron spacer ring on top of the last sapphire disc, and use filter paper to remove any excess liquid. While holding the samples, make sure you do not trap any air bubbles between the sapphire discs. Then place the entire assembly between the two transparent half cylinders and close the top red cover to initiate the freezing process.
The red cover will pop back up automatically once the freezing process is complete. When all of the samples have been frozen, open the door to the storage dewar and transfer the dewar to the benchtop. Transfer the specimen chamber from the storage dewar to a specialized sample tray filled with liquid nitrogen.
And unlock the knob to release the specimen cup. Then use a pair of tweezers with liquid nitrogen cooled tips to remove the transparent half cylinders from the specimen cup. And carefully transfer the middle black plates to a small cup containing liquid nitrogen.
Take care to keep the frozen samples under liquid nitrogen at all times, and to keep always handle the samples with pre-cooled tweezers. Pre-cool the tweezers again, and quickly transfer the first middle plate to the pre-cooled acetone. Gently shaking and tapping the plate to separate the sapphire disc from the plate.
Then place an open cryogenic file of fixative into a specimen chamber in the substitution unit. Transfer the sapphire disc into the vial. And immediately cap the vial.
When all of the samples have been transferred, set the appropriate free substitution program parameters and start the program. At the end of the free substitution program, use gloves to transfer the cryogenic files from the specimen chamber to a chemical hood and use a pipette to add room temperature acetone to each vial. Replace the wash in each vial with fresh acetone four to six times over a one to two hour period.
Then add freshly prepared 30%epoxy resin to each vial for a two to three hour incubation at room temperature in an orbital shaker at 120 rpm. At the end of the incubation, replace the 30%epoxy resin with 70%epoxy resin for a three to four hour incubation on the orbital shaker. Then use tweezers to transfer each disc, cell side up, to the cap of an embedding capsule and add 90%epoxy resin to the discs for an overnight incubation at four degrees Celsius.
The next morning, transfer each sapphire disc to the cap of a new embedding capsule, and fill the cap with freshly prepared 100%epoxy resin. After the last 100%epoxy resin incubation, polymerize the samples in a 60 degree Celsius oven for 48 hours. When the samples have polymerized, place the first sample upside down on the stereo microscope so that the sapphire disc is located at the top of the resin block, and use a razor blade to remove a thin layer of resin from the top of the sample.
Next, use the razor blade to cut a shallow line along the edge of the disc to separate the sapphire disc from the epoxy resin. And dip the disc into liquid nitrogen for about 10 seconds to separate the disc from the rest of the resin. Transfer the resin under a dissecting microscope, and tape the sample to the microscope stage.
Using a four to 10X magnification, find an area with cells and use a double edged razor blade to cut a two by two millimeter area around the region of interest. Then use superglue to mount the cells onto a cylindrical dummy block made of epoxy resin, and incubate the block at 60 degrees Celsius for one hour. After sectioning on an ultra micro tome, transfer the samples to a transmission electron microscope for imaging at a 930, 000X magnification.
In this image, synaptic vesicle fusion via exocytosis recorded 15 milliseconds after light onset within the active zone of a presynaptic membrane in a mouse hippocampal neuron is shown. 100 milliseconds after light onset an endocytotic pit adjacent to the active zone can be observed, illustrating ultra fast endocytosis by the nerve cell. After watching this video you should have a good understanding of how to process samples with flash and freeze experiments.
Remember, working with glutaraldehyde, osmium tetroxide, and liquid nitrogen can be really dangerous, and so make sure you wear protective clothing. Once mastered, the freezing portion of this procedures can be done in a couple hours for 12 samples, but the rest of the procedures including imaging and image analysis can take up to days or weeks or even months, and so it could be a really long procedures. If you localize proteins, or visualize their dynamics, you can also use different protocols to preserve antigenicity, or pearlescence from pearlescent proteins and visualize their dynamics using these techniques.
