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.
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Cut
the specimen into small pieces, approximately 1mm x 1mm
x 3mm. Note: Short wide mouth vials are excellent receptacles
for tissue processing.
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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.
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Samples may be left overnight
as required.
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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.
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* Choice of fixative
and concentration varies for different tissue types. |
2.Cryo-protection.
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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%.
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Dilute the glycerol in the
same buffer as that used in the fixation step.
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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
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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.
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Secure the rivet into a suitable
holder and plunge into liquid nitrogen slush. |
4. Cryo-Preparation Chamber
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Transfer
under vacuum into the cryo- preparation chamber and fracture. |
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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. |
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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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Gatan
Inc. Corporate Headquarters, 5933 Coronado Lane, Pleasanton,
CA 94588
Tel. (925) 463 0200 Fax. (925) 463 0204
Contact: info@gatan.com
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