Electron microscope

1. Mr.Mahmoud Ibrahim

2. 

3. 

4. 

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.

9. 
Post mortem changes and fixation
artefact reduced with small samples.

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

18. 
The dehydrating times should be
adjusted to size and kind of tissue.

19. 
Optimum impregnation as
follows:
First place tissue in a solution
composed of equal volume from
propylene oxide and resin.
Impregnation

20. 
Second transfer tissue to 25:75
propylene oxide and resin.
Finally put tissue in pure resin.
An hour in each step is adequate.

21. 
Periodic agitation is
recommended in each step.
Incomplete impregnation causes
major sectioning difficulties.

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.

27. 
5- Stable in E.M.
6- Easy section.
7- Available.

28. 
Epoxy resin composed of:
1- Monomeric resin.
2- Hardener.
3- Accelerator.
4- Plasticizer.

29. 
Hardness, flexibility and
polymerization times can be
manipulated by varying the
amount of individual
components

30. 
The prepared resin is best
delivered through a non-
reactive plastic syringe or
pipette.

31. 
The widely used epoxy resins
are:
1- Araldite.
2- Epon.
3- Spurr,s resin.

32. 
The exposure to epoxy resins
causes allergic contact dermatitis.
May be carcinogenic.
Toxic.
Irritants.

33. 
Derive from methacrylic acid
and acrylic acid.
Used to infiltration and
embedding as epoxy resins.
Acrylic resins (methacrylates)

34. 
Polymerization obtained by:
light, heat or chemical
accelerator at room temperature

35. 
The most common acrylic resins
are:
LR white, LR gold and the
Lowicryl series (K4M, K11M,
HM20 and HM23).

36. 
Manual tissue processing is
preferably for E.M.
Cell suspensions can be
demonstrated by E.M.
Tissue processing

37. 
Glass or diamond knives used.
Higher angle knives up to 550 for
hard tissue while 350 for soft
tissue.
Ultra microtomy

38. 
D.W or deionized water used.
10-15% of ethanol or acetone can
also be used but not with a
diamond knife.
Floatation

39. 
The floatation fluid should be
attached to the knife

40. 
Can be achieved manually or by
using ultra microtome.
Trimming

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

42. 
Semi-thin sections usually
measured between 0.5-1
micrometer.

43. 
Usual section thickness 0.1-
0.005 micrometers.
Optimum section thickness
measures 80 nm.
Ultra-thin sectioning

44. 
To collect sections immerse the
grid in the floatation fluid, then
placed the ribbon over the grid.

45. 
After sections collection, the
grids transferred by using forceps
to filter paper on a Petri dish, and
allow to dry completely before
staining.

46. 
Achieved by introducing heavy
metal atoms which deposit on
the tissue components.
Staining

47. 
Tissues are stained at several
points during preparation:
1- During secondary fixation.

48. 
2- When uranyl acetate is used
during the post fixation wash.
3- By staining the sections with
lead and uranium salts.