The overall goal of the following experiment is to immobilize the cellular structures of plant cells by high pressure, freezing and quick freeze substitution for subsequent visualization by transmission electron microscopy. This is achieved by carefully preparing a tissue sample for freezing by coding it with a cryoprotectant to ensure that the tissue is minimally damaged during high pressure freezing. As a second step, the sample is rapidly frozen under high pressure, which immobilizes the cell components and limits formation of crystalline ice, resulting in cells with intact morphology.
Next quick free substitution is performed in order to replace the cellular water with organic solvents and to introduce fixatives such as osmium tetroxide, thus linking and stabilizing cellular ultra structures. Results are obtained that show high pressure freezing and quick freeze substitution effectively preserve plant cellular structures and morphology based on transmission electron microscopy. The Main advantage of this technique over existing methods like conventional chemical fixation is that H-P-F-Q-F-S immobilizes cellular contents in millisecond times minimizing artifact formation.
Generally, the QFS protocol is challenging for a beginner, so it is recommended that two people work together until users are comfortable with the procedure. Personal protective equipment, including cryo gloves and goggles, must always be worn when handling liquid Nitrogen fill an insulated box containing cryo vial holders with liquid nitrogen so that the holders are completely covered. The fixative use during quick freeze substitution or QFS is 1%osmium, tetroxide, and 0.1%urinal acetate in acetone.
After preparing a large volume of the solution following all safety warnings, aliquots of 1.5 milliliters are dispensed into cryo vials and stored frozen in liquid nitrogen. Place the appropriate number of labeled cryo vials containing freeze substitution or FS medium into the aluminum, two holders in the liquid nitrogen. With this protocol up to four discs each containing a single sample can be placed in a single vial about one to one and a half hours prior to sample preparation.
The high pressure freezer must be switched on and prepared for the run. This protocol also requires yeast paste as an extracellular cryoprotectant for filling the space around the sample. In the specimen carrier mix some baker's yeast with a roughly equal volume of 10%methanol using a toothpick until the paste is smooth.
With a consistency of pancake mix, the amount of paste needed depends on the number of samples for 10 samples or fewer, the paste can be mixed in a micro centrifuge tube. To prepare a sample for high pressure freezing or HPF, remove a leaf of interest from the plant and gently place it on a piece of dental wax or other cutting surface. Use a 0.2 millimeter punch to cut a sample out of the leaf.
The most critical part of this procedure is this sample preparation for loading into this specimen carrier. If the sample is poorly handled, no other step will redeem the damage cost Working under a dissecting microscope. Find the 0.2 millimeter side of the type A specimen carrier and coat it with yeast paste.
Place the leaf disc in the specimen carrier, smooth out the paste with a fine paintbrush to ensure that the disc is completely filled and the paste is level with the rim of the holder. Place the specimen carrier in the specimen holder, which should be dry and at room temperature, cover the sample with a type B specimen carrier flat surface down. Place the insulated box that was prepared earlier on top of the machine.
Take the sample in the specimen holder to the high pressure freezer. Insert the specimen holder containing the sample into the high pressure freezer. Initiate a freezing cycle by pressing the jet auto button, which should complete in a second or two working as quickly as possible.
Remove the specimen holder from the machine and place the tip. Holder the sample into the liquid nitrogen in the insulated box. Immerse the tips of two pairs of forceps in the liquid nitrogen to chill them after freezing.
The carriers should only be handled with liquid nitrogen cooled forceps working under the liquid nitrogen. Open the specimen holder by turning it anti-clockwise and remove the specimen carrier from the holder with liquid nitrogen cooled forceps, ensuring that the disc is always in the liquid nitrogen or vapor. Open a cryo vial containing FS media and place the lid on the side of the box.
Hold the cryo vial with one pre cooled forceps and use the other forceps to place the disc in the cryo vial. Screw the lid of the cryo vial back on being careful not to trap any liquid nitrogen in the vial. Liquid nitrogen expands 700 fold during warming and any trapped liquid nitrogen can cause explosions during this time.
Repeat this process until all desired samples are frozen. Multiple leaf discs containing the same type of sample can be placed in the same vial. To begin the preparations for free substitution completely immerse an aluminum heater block in liquid nitrogen for 10 minutes or until nucleate boiling stops.
In the meantime, place a layer of dry ice at the bottom of the container to be used as a QFS chamber. A styrofoam container or ice bucket with crushed dry ice or pellets can be used. A temperature probe is needed for this procedure.
A thermocouple is placed through the top of a cryo vial so that it reaches the bottom of the tube and the lid of the tube is sealed with resin so that no liquid leaks out. Fill the tube with 1.5 milliliters of acetone at the beginning of each QFS run. Be sure that the lids on the vials are tightly screwed on so that the FS media does not leak out during fs.
Quickly insert the cryo vials containing the samples along with the temperature probe into the middle rows of the heater block. Begin recording the temperature using insulated cryo gloves or large forceps. Pour out all the liquid nitrogen from the heater block.
Take care not to pour out the cryo vials. Place the block containing the vials into the layer of dry ice in the QFC chamber. Make sure the block is placed so that the tubes are lying horizontally, but with a slight upward tilt.
There should be no leakage if the correct cryo vials are used. Pack the QFS chamber with dry ice so that the FS tubes are covered. Although it is not necessary to cover the top of the block, place the lid on the chamber.
The QFS must be performed in a fume hood. Placed a QFS chamber with the samples on a platform rotary shaker and rotate at 125 RPM for 120 minutes. During this time, the temperature of the block should gradually increase to about negative 80 degrees Celsius.
The agitation ensures mixing of the components for better fs. After two hours, remove the dry ice from the chamber. Use a cryo glove to swiftly lift the heater block out of the QFS chamber and quickly pour out the dry ice into a secondary container.
Continue shaking for another hour. During this time, the temperature should increase to about negative 15 degrees Celsius to negative 20 degrees Celsius. At the end of the hour, remove the samples and temperature probe from the QFS chamber and place them on the shaker at room temperature for another 10 to 15 minutes until they reach room temperature.
Stop recording the temperature when the quick free substitution is done. Carefully remove the FS media from each cryo vial with a plastic transfer pipette and place in the appropriate container for toxic waste. Wash the samples four times with 100%acetone.
Collect the first two washes and place in the toxic waste container. Take care not to discard the samples when removing the acetone. After the samples have been washed, use fine forceps to remove the tissue samples from the specimen carrier.
Keep samples wet with acetone as this is done and work very gently to avoid breaking the samples. It is not unusual to have the yeast paste fall away from the samples. At this point, the yeast paste is usually a very dark brown while the leaf tissue is still green.
Often the tissue samples fall out of the specimen carrier during the FS collect the samples using a transfer pipette in cryo vials containing acetone. Subsequently, the samples are prepared for transmission electron microscopy according to standard protocols. A.A typical curve for temperature changes during QFS is shown in this graph.
The temperature increases rapidly from negative 196 degrees Celsius, the temperature of liquid nitrogen to about negative 80 degrees Celsius. The spike at the beginning of the run is due to the electronics of the probe and does not reflect the real temperature measurement after HPF and QFS samples are prepared for viewing by transmission electron microscopy. Typical results are shown in these images from a rabbit opsis leaf samples.
Plasma membranes that are smooth and pressed against the cell wall indicate good fixation. A branched plasma dema is visible at high magnification. Other organelles are also clearly visible.
They include chloroplasts and the thylakoids mitochondria, GOGI microtubules, and plasmas desa, the large central VAEs remain intact. Poor handling during H-P-F-Q-F-S results in artifacts including ice crystal induced damage and lysis. Lead precipitate may also form during staining of sections Once mastered.
This technique can be done in four and a half hours if it is performed properly. Don't forget that working with liquid nitrogen and osmium tetroxide can be extremely hazardous and precautions including working in the fume hood and wearing personal protective equipment, including gloves, lab coats and goggles should be taken while performing this procedure.
