Biol/Vpat/Cellbio 5040/7040 Electron Microscopy
Lecture for 8/22/03
Fixation of Biological Tissues (Refer to Chapter 2 in Bozzola)
A. Brief Review of TEM/SEM operation to show why we prepare the sample.
Two factors (conditions required within the scope) affect the sample in the scope:
1. The electron beam is of sufficient strength to pass through the sample (Heat or beam damage)
2. The high vacuum required to allow for a "mean free path" of electrons (Dessication)
A high vacuum will have several benefits:
a. The vacuum needs to be high to allow the electrons to have a "mean free path" (10-4 Pa or ~10-6 torr to travel far enough in TEM). The focused electrons need to either strike the sample and, in TEM, pass through it and reach the screen.
b. Protects the filament from oxidation.
c. removal of contaminants (water vapor, outgassing of volatile organics) that are released when in vacuum or interacting with intense electron radiation.
Problems with intense radiation from beam?
Sample distortion/damage , outgassing.
In TEM, the electrons will be passing through the sample – must be thin enough (30-100 nm thick).
Problems associated with cutting samples that thin
- how to cut?
- holding the sample in the scope?
- Beam damage when that thin
- drying in the vacuum
- outgassing of components
We correct this by fixing the sample and embedding in a beam/vacuum stable resin.
TISSUE PREPARATION – Overview (overhead of Prep flow chart)
With both SEM and TEM, we want to keep the tissue or organism or sample from changing.
- Primary Fix (usually with aldehyde)
- Second Fix (usually with OsO4)
- En-bloc staining with electron scattering chemical (e.g. uranyl acetate or tannic acid, etc..) (optional)
- Dehydration with ethanol series to 100% (replacing the water)
- Transition from EtOH to resin-miscible solvent (either propylene oxide or acetone) ensures good polymerization
- Gradual infiltration of the sample with non-polymerized resin
- Polymerization of resin-embedded sample
- Primary Fix (either aldehyde or OsO4)
- Secondary Fix (optional if primary fix is OsO4)
- Dehydration with EtOH to 100% (replace water)
- Critical point dry (changing EtOH to liquid CO2 to gaseous CO2)
- Mount and coat with conductive material