Gatan, Inc. How to prepare high water content bulk biological and botanical specimens for Cryo-SEM and not get ice crystals.

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.

Figure 1. Quenched cooled renal tissue. Scale bar = 1 micron. (Click on image for a larger copy)

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.

Figure 2. Glycerol protected renal tissue. Compare this figure to figure 1 above. Scale bar = 1micron. (Click on image for a larger copy)

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:

Figure 3. Cryo-protected renal tissue. A cell in a collecting duct of a uriniferous tubule. Numerous nuclear pores are shown en face and surface. Both inner and outer membranes of the nuclear envelope are also evident. (Click on image for a larger copy)

Figure 4. Cryo-protected rodent intestine epithelial cell. Microvilli and tight intercellular junction illustrated. Scale bar =100nm. (Click on image for a larger copy)

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.

Figure 4*. Cryo-protected sublimated rodent heart muscle. Image courtesy of Dr Cameron Ackerley, Sick Children’s Hospital, Toronto, Canada. (Click on image for a larger copy)

To Prepare a Specimen:

1. Fixation.

	•	Cut the specimen into small pieces, approximately 1mm x 1mm x 3mm. Note: Short wide mouth vials are excellent receptacles for tissue processing.
	•	Quickly immerse the small pieces of tissue into a suitably buffered glutaraldehyde* solution or a freshly prepared para-formaldehyde and glutaraldehyde combination and leave to infuse at +4C.
	•	Samples may be left overnight as required.
	•	In some cases, where facilities are available, it may be more desirable to undertake in situ or perfusion-fixation, and then dissect into smaller segments.
	* Choice of fixative and concentration varies for different tissue types.

2.Cryo-protection.

	•	Wash off the residual fixative well and transfer into (in this example) glycerol. Depending on requirements this may be at a concentration between 10 to 20%.
	•	Dilute the glycerol in the same buffer as that used in the fixation step.
	•	Raise the concentration gradually from 5% through to 20% in 10 min intervals.

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

	•	Place a small cut piece of cryo-protected tissue into a copper rivet as provided with your Alto system, so that a portion protrudes from the top.
	•	Secure the rivet into a suitable holder and plunge into liquid nitrogen slush.

4. Cryo-Preparation Chamber

	•	Transfer under vacuum into the cryo- preparation chamber and fracture.
	•	Depending on type and final concentration of cryo-protectant used the sample may be sublimated. A sublimation temperature of -100C for 3mins is suggested for specimens protected with 15% glycerol. However, these parameters will depend on the vacuum conditions prevailing at the time, so some adjustment may be required.
	•	Following fracturing/sublimation, coat the specimen and image.

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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