5.
TEM is a microscopy technique
whereby a beam of electrons is
transmitted through an ultrathin
specimen, interacting with the
specimen as it passes through it.
Introduction
6.
An image is formed from the electrons
transmitted through the specimen,
magnified and focused by an objective
lens and appears on an imaging screen.
7.
The standard approach is to immerse
the specimen in fixative pre-cooled to
4oc immediately after collection.
Specimen handling
8.
Once in the fixative the specimen is
selected using scalpel, then transferred
to glass vial.
The final specimen size is usually
1mm3.
10.
The standard fixation protocol involves
primary fixation with glutaraldehyde at
40c followed by secondary fixation in
osmium tetroxide.
Fixation
11.
Glutaraldehyde is effective at a
concentration 1.5-4%, while osmium
tetroxide optimum concentration 1 or
2%.
Fixation concentration
12.
Optimum temperature 4oc.
Fixation at room temperature improve
the penetration rate, reduce the time but
increases the risk of autolytic change.
Fixation temperature
13.
For 0.5-1mm3 tissue specimen in
aldehyde fixatives 4-6 hrs.
Secondary fixation in osmium tetroxide
required 60-90 minutes.
Fixation duration
14.
Optimum pH ranged between 7.2-7.6.
Inadequate buffering can lead to
significant loss of cellular components,
with subsequent shrinkage or swelling.
Fixation pH
15.
Generally is not a major requirement.
If necessary 300-330 mOsm.
Osmolality equivalent to that of plasma
or slightly hypertonic.
Fixation osmolality
16.
Rinsing the tissue in buffer after post
fixation.
Done to remove surplus fixative and
provide an opportunity for tissue storage
if necessary.
Wash buffer
17.
Dehydration usually carried out in
ethanol with the combination of
propylene oxide to facilitate resin
infiltration.
Dehydration
22.
Tissue samples are placed in an
appropriate mold (capsules
made from polyethylene glycol)
filled with resin and allowed to
polymerized using heat.
Embedding
23.
A paper strip bearing the tissue
identification code written in
pencil should be used.
Flat embedding molds made from
silicone can be used.
24.
The most common embedding media
for E.M are:
1- Epoxy resins.
2- Acrylic resins.
Embedding media
25.
Characterized by:
1- Contains oxygen and 2 carbon
atoms (epoxide), cross linking
between them creates a three
dimensional polymer of great
mechanical strength.
Epoxy resins
26.
2- With little shrinkage (less
than 2%).
3- Permanent.
4- Preserve tissue ultra structure.
41.
Allow samples to be screened
for specific features and to
select areas for thin sectioning.
Carried out by using glass not
diamond knife.
Semi-thin section