1. TRANSMISSION ELECTRON
MICROSCOPE

2. TRANSMISSION ELECTRON
MICROSCOPE
 The first TEM was built by Max Knoll and Ernst
Ruska in 1931, with this group developing the
first TEM with resolution greater than that of
light in 1933 and the first commercial TEM in
1939.
 It is capable of imaging at a significantly
higher resolution than light microscopes, owing
to the small wavelength of electrons.
 TEM is far more useful for medical investigations
than SEM
 TEM forms a major analysis method in a range of
scientific fields, in both physical and biological
sciences.
Cancer research, virology, materials science as
well as pollution , nanotechnology,
and semiconductor research.

3.  Electrons transmitted through the specimen are
focused and the image magnified by using
electromagnetic lenses (rather than glass lenses)
to bend the trajectories of the charged electrons.
 Image is focused onto a viewing screen or film.
 Used to study internal cellular ultrastructure

4. STRUCTURE

5. STRUCTURE

6. PARTS OF TEM
Vacuum system
 To increase the mean free path of the electron
gas interaction
 allowance for the voltage difference between the
cathode and the ground without generating an
arc, and secondly to reduce the collision
frequency of electrons with gas atoms to
negligible levels

7. Specimen stage
 to allow for insertion of the specimen holder into the
vacuum with minimal increase in pressure in other
areas of the microscope
Electron lens
 are designed to act in a manner emulating that of an
optical lens, by focusing parallel rays at some constant
focal length

8. Apertures
 annular metallic plates, through which electrons
that are further than a fixed distance from
the optic axis may be excluded

9. HOW TO VIEW SLIDES UNDER
TRANSMISSION ELECTRON
MICROSCOPE
 The imaging systems of TEM consist of a phosphor
screen, which may be made of fine (10–100 μm)
particulate zinc sulphide, for direct observation by the
operator. Optionally, an image recording system such
as film based or doped YAG screen coupled
CCDs.Typically these devices can be removed or
inserted into the beam path by the operator as
required.

10. OPTICS
condensor lenses
 responsible for primary beam formation
objective lense
 focus the beam that comes through the sample
itself
projector lenses
 used to expand the beam onto the phosphor
screen or other imaging device, such as film.

11. PRINCIPLES OF TEM
 Illumination - Source is a beam of high velocity
electrons accelerated under vacuum, focused by
condenser lens (electromagnetic bending of
electron beam) onto specimen.
 Image formation - Loss and scattering of
electrons by individual parts of the specimen.
Emergent electron beam is focused by objective
lens. Final image forms on a fluorescent screen
for viewing

12.  Image capture – on negative or by digital camera

13. FIXATION OF TISSUES FOR EM
 Must be prompt
 Cut to 1-2 mm cubes
 Use sharp razor blade, avoid crushing
 2.5% glutaraldehyde for 4 to 12 hours
 Postfixation in 1% osmium tetroxide

14. TISSUE PREPARATION FOR TEM
 Dehydration in alcohol
 Embedding in resin
 Semithin sections cut at 0.5 micron thick, stained
with toluidine blue
 Selection of sample blocks
 Ultrathin sections at 0.1 micron thick, stained
with lead citrate and uranium acetate

15. SEMITHIN SECTIONS
STAINED WITH TOLUIDINE BLUE

16. ULTRATHIN SECTIONS ON GRID

18. HOW ARE ELECTRONS EXCITED
 When an electron beam passes through a thin-
section specimen of a material, electrons are
scattered.

20. ADVANTAGES OF TEM
 high magnification at high resolution
 technique largely standardized
 some ultrastructural features are highly specific
for certain cell types or diseases

21. DISADVANTAGES OF TEM
 equipment is expensive
 procedures time consuming (staff costly)
 small samples lead to possible sampling error
and misinterpretation
 optimum tissue preservation requires special
fixative and processing
 much experience is needed for interpreting the
results
 Time consuming
 Works in the dark
 Photography required