How to prepare high water content bulk biological and botanical specimens for Cryo-SEM and not get ice crystals.
By Marilyn Carey, Cryo-SEM Product Specialist, Gatan UK.
One of the problems encountered with cryogenic electron microscopy is the formation of ice crystals when hydrated specimens are quench cooled. These crystals can be big; especially when the sample is large e.g. 0.5mm in size and the sample contains mostly water, as found in biological tissue. The ice crystals that form damage the internal microstructure of cells. Cytoplasm in particular demonstrates this effect well; large ice crystals with eutectic boundaries are well recognised features in cells of tissue that has been quench cooled prior to observation in the cryo-scanning electron microscope, as seen in figure 1.
To reduce ice crystal formation during sample preparation an efficient rapid cooling procedure is necessary e.g. high pressure freezing. However, this instrumentation is prohibitally expensive for many, and (at best) only the first 200 microns of the specimen has negligible ice damage. Consequently for bulk specimens this approach is often not an option. To overcome these problems precautions must be taken to inhibit the formation of ice crystal segregation zones and cryo-protective agents are employed. Good cryo-protective agents are those that are non-toxic, have highly permeability for cells, have the ability to reduce volume expansion during freezing and have minimal cryo-protectant induced structural distortion. Commonly employed penetrating cryo-protectants include glycerol and DMSO (either in combination or separately), raffinose, sucrose and glucose. These cryo-protective agents serve to retard the formation of ice crystals by lowering the temperature of homogeneous ice nucleation and thereby facilitating the formation of a vitrified (glass like) ice phase as shown in figure 2.
This phase serves to maintain the interaction between components and stabilizes diffusible elements. It should be remembered however that selection of suitable cryo-protective agent(s) and its applied concentration is dependant on the desired outcome as well as the nature of the specimen.
To improve permeability of the cryo-protectant into intracellular compartments of cells mild chemical fixation is commonly employed. The fixative provides two necessary functions, firstly it makes the cells competent for the subsequent step i.e. cryo-protection, secondly, it enables the removal of soluble material which might otherwise obscure detail.
Some more examples:
Generally, samples prepared using cryo-protectants cannot be sublimated. This is because the agents are non-volatile. However, if the concentration of the agent is such, some sublimation may be possible, enabling exposure of otherwise obscured structures, see figure 4.
To Prepare a Specimen:
It should be noted that the exact time for complete infiltration will depend on sample type, and importantly on the cryo-protectant used and its possible damaging effects.
3. Sample Mounting and Cooling
4. Cryo-Preparation Chamber
Depending on the length of the specimen
protruding from the rivet it may be possible and desirable
to re-fracture the specimen to provide additional information.
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