The overall goal of this protocol is providing us with electromicroscopy resolution of the water distribution in various kinds of xylem cells in situ. This method has been used to observe the water in xylem in order to clarify water distribution and the changing water regimes, the seasonal variation of water distribution, the effect of freeze/thaw cycles, the distribution of water in wet wood, and cavitation induced by certain biotic stresses. Freeze-fixation of a living trunk under high hydraulic tensions sometimes causes artificial cavitations, which are observed by cryo-SEM as ruptured ice crystals in the lumen of conduit.
This updated procedure focuses on obtaining high-quality aerotome micrographs of xylem without the artifact in the sampling procedure. Viable demonstration is critical because there are a number of important details that cannot be easily explained. At the same time, obtaining training is not easy because only a few laboratories around the world practice this method.
To begin this procedure, enclose a branch and leaves for sampling with a black plastic bag to equilibrate the water potential between xylem and leaves more than two hours before sampling. Using a pressure chamber, determine the water potential of at least two leaves from the sample. When the water potential is higher than approximately minus 0.5 megapascals, a sample can be harvested after freezing.
When the water potential is lower than minus 0.5 megapascals, proceed with the treatment for relaxation. Fix a watertight collar around the stem, which will be filled with water. Using pruning shears or a saw, cut the stem under the water surface and keep the cut end of the sample underwater.
Transfer the sample to another container of water as quickly as possible to minimize exposing the cut end to air. For broad-leaved species, ensure that the length from the spot where cryo sample for SEM will be obtained to the cut edge of the harvested stem is longer than the sample's maximum vessel length in order to prevent tension-induced artifacts within the crysoample. Next, cover the sample with a black plastic bag to reduce transpiration.
Keep the cut end of the sample in the water and maintain this condition for approximately 30 minutes in order to relax the xylem tension. After this, measure the water potential again to confirm the relaxation of the xylem tension. First, use scissors or a utility knife to cut and open one side of a water-tight collar.
Attach the collar tightly around the stem with adhesive tape while holding the aperture horizontally. Put on insulating gloves, and safely hold the vessel of liquid nitrogen. Run liquid nitrogen into the collar until it is full and keep it filled by steadily adding additional liquid nitrogen to completely freeze the water in the xylem.
After sufficient freezing time, detach the collar from the frozen portion of the sample stem in order to remove the liquid nitrogen. Immediately use a fine hand saw to harvest the sample. Then cover the frozen sample with a piece of aluminum foil or put it back into a sample tube.
Rapidly place the harvested sample into a container filled with liquid nitrogen. Store the samples at minus 80 degrees Celsius until ready to perform the observation. To begin, set the temperature of the specimen chamber of the cryostat to minus 30 degrees Celsius, which is usually cold enough to keep the xylem sap of most plants in a frozen state.
Use a sharp knife or a fine-toothed saw to trim the sample into a small piece that can be adjusted for the specimen holder of a cryo-SEM. Mount the trimmed piece to a chuck, a holder for a cryostat, with tissue-freezing embedding medium for cryo-sectioning. Attach the chuck to a specimen holder of a microtome of the cryostat.
Trim the surface by repeatedly shaving off five to seven micrometer thick sections. Cutting away more than 1, 000 to 2, 000 micrometers in total depth to the initial surface is useful for elmininating the damaged portion of the sample caused by pre-cutting. After roughly trimming a surface of the sample, adjust an unused portion of the microtome blade above the specimen's surface.
Slightly widen the distance between the surface of the specimen and the blade. And cut the surface only once or twice. Then, slide the blade again and position an unused portion of the blade onto the specimen surface.
Repeat this cutting processing three to four times to obtain a clear surface without knife marks. After the final cut, set the blade's position far from the sample to prevent dust from sticking onto the sample. And detach the chuck from the microtome.
Next, attach the specimen to a cryo-SEM specimen holder with tissue-freezing embedding medium in the cryostat chamber. First, use liquid nitrogen to maintain a temperature under minus 120 Celsius in the cryo-SEM specimen chamber according to the equipment user's manual. Next, place the specimen holder with the prepared specimen into an insulating container filled with liquid nitrogen in the cryostat chamber.
Use a specimen exchanging rod to hold the specimen holder beneath the liquid nitrogen. Rapidly transfer the specimen holder to the pre-evacuation chamber of the cryo-SEM specimen chamber as soon as starting evacuation of the pre-evacuation chamber. To begin, turn on the acceleration voltage of the electric gun.
Next, raise the temperature of the specimen stage to minus 100 degrees Celsius. Wait several minutes for the frost dust to be removed and for the surface level of the ice in the xylem cells to decrease slightly compared to the cell walls. Then, lower the temperature of the speiumen stage to minus 120 degrees Celsius.
In this study, cryo-SEM observation methods are used to clearly visualize water distribution on a cellular scale. At low magnification, the black area in the images indicates the cavities from which water entirely or partly disappears, while the gray area indicates xylem cell walls, cytoplasm and water. At high magnification, it becomes apparent that the water is not entirely lost from the lumina of three tracheids, indicating the occurrence of macro bubbles in the xylem sap in situ.
With respect to broad-leaved species, cavitation occurrence is easily detected within vessels, while water existence is hard to distinguish within fibers, especially at low magnification. Cytoplasm and parenchyma cells can be distinguished from water within tracheids or vessels through ice plane textures. Analysis of the effect of temperature on the freeze-etching process reveals that frost dust is gradually sublimated, and endotracehid pit membranes become clearer through the progression of sublimation with increasing temperature.
Remaining large frost dust particles can be eliminated by further freeze-etching. But this can be problematic as it unnecessarily decreases the surface level of ice in xylem conduits. The high-quality of observation is largely achieved through accurate specimen preparation.
The smoothing of the surface with the sharp blade of the microtome is especially important. Insufficient smoothing by a used blade can sometimes create a rough surface that resembles knife marks, or can create numerous occurrences of dust from the cuts. Sample freezing without the relaxation of negative water column pressure will cause artifactual induction of cavitation in xylem conduits.
Clustered ice crystals are observed in vessels of specimens where the sample was not relaxed. Contrastingly, no clustered ice crystals are observed in the relaxed sample specimens with a similar water potential. The application of freeze-fixation to transpiring living plants who are suffering drought may induce result-based artifacts such as clustered ice crystals.
Those artifacts lead to misinterpretations of the water status in xylem conduit. So it is important to first confirm the water potential of samples before freeze-fixation. Although this procedure provides for an image of what status in xylem conduit, a living tree must be destroyed for the observation.
Combining other non-invasive observation methods such as MRI or micro-CT with subsidiary level image observation will deepen our understanding of the nature between water transport and usage. The observation of water status introduced by this paper will also provide a method for the clarification of the relationship of water that is mixed in cells and other anatomical responses to abiotic or biotic stress. Cryo-SEM equipped with energy dispersive x-ray spectroscopy or top sim have been used to study element distribution over the surface of a specimen containing water.
Combining elemental analysis and this procedure in that similar frame can give us profound knowledge of the xylem cell behavior related to water status. When using liquid nitrogen for freeze-fixation of samples in a room, do not forget to ventilate the room to avoid oxygen deficiency.
