The document provides an overview of transmission electron microscopy (TEM). It discusses how TEM works, the various components of a TEM, sample preparation techniques including fixation, dehydration and embedding, and imaging modes such as negative staining and shadow casting. TEM allows visualization of structures at the nanoscale and provides greater magnification than light microscopy. Proper sample preparation is crucial to obtain high quality images.

1. BACHELOR OF SECONDARY EDUCATION
CENTRAL BICOL STATE UNIVERSITY OF
AGRICULTURE

2. INTRODUCTION
Electron Microscopy involves the study of different
specimens by using an electron microscope.
 Electron microscopes are scientific instruments
that use a beam of energetic electrons to examine
objects on a very fine scale.
Through the use of an electron microscope we can see
things that we would not normally be able to see with
our naked eyes and has greater magnification than
light microscope.
 This required 10,000x plus magnification which was
not possible using current optical microscopes

4. TYPES OF ELECTRON
MICROSCOPE
1. TRANSMISSION ELECTRON MICROSCOPE-
form images using electrons that are transmitted
through a specimen.
2. SCANNING ELECTRON MICROSCOPE-
utilize electron that have bounced off the surface
of the specimen.

5. The transmission electron microscope (TEM) was the first type
of Electron Microscope to be developed and is patterned exactly on
the light transmission microscope except that a focused beam of
electrons is used instead of light to "see through" the specimen. It
was developed by Max Knoll and Ernst Ruska in Germany in 1931.

6. TRANSMISSION ELECTRON
MICROSCOPY (TEM)
 Transmission electron microscopy (TEM) is a
microscopy technique whereby a beam of electrons is
transmitted through an ultra thin specimen, interacting
with the specimen as it passes through. An image is formed
from the interaction of the electrons transmitted through
the specimen; the image is magnified and focused onto an
imaging device, such as a fluorescent screen, on a layer of
photographic film, or to be detected by a sensor such as a
CCD camera.

7. DIFFERENCES BETWEEN OM AND EM
OPTICAL MICROSCOPE ELECTRON MICROSCOPE
1. The source of light. 1. The light source is replaced by a beam of
2. The specimen. very fast moving electrons.
3. The lenses that makes the 2. The specimen usually has to be specially
specimen seem bigger. prepared and held inside a vacuum
4. The magnified image of the chamber from which the air has been
specimen that you see. pumped out (because electrons do not
travel very far in air).
3. The lenses are replaced by a series of coil-
shaped electromagnets through which
the electron beam travels.
4. The image is formed as a photograph
(called an electron micrograph) or as an
image on a TV screen.

8. MICROSCOPE RESOLUTION MAGNIFICATION
OPTICAL 200 nm 1000x
TEM 0.2 nm 500000x

10. DIAGRAM TO REPRESENT TEM’S WORKING
Virtual Source
First Condenser Lens
Second Condenser Lens
Condenser Aperture
Sample
Objective Lens
Objective Aperture
Selected Area Aperture
First Intermediate Lens
Second Intermediate Lens
Projector Lens
Main Screen (Phosphor)

11. DIFFERENT COMPONENTS OF TEM
1. HIGH TENSION CABLE
2. ELECTRON EMITTER
3. STEPPER MOTORS FOR
CENTERING THE ELECTRON BEAM
4. CONDENSER 11. VACUUM PUMP LEADS
5. APERTURE CONTROLS
12. GONIOMETER
6. SPECIMEN HOLDER
13. VACUUM AND
7. OBJECTIVE LENS
MAGNIFICATION CONTROL
8. PROJECTOR LENS
14. FOCUSING CONTROL
9. OPTICAL LENS
10. FLUORESCENT SCREEN

12. SOME TYPICAL TEM MODELS

13. TEM SAMPLE PREPARATION
 Cleaning the surface of the specimen
The proper cleaning of the surface of the sample is
important because the surface can contain a variety of
unwanted deposits, such as dust, silt, and detritus,
media components, or other contaminants.
The best way to clean the surface of specimen from
contaminants is to carefully rinse them three times for
10 min in 0.1 M cacodylic acid buffer (pH 7.3) at room
temperature.

14. TEM SAMPLE PREPARATION
 Primary fixation of the specimen
Fixation can be achieved by perfusion and
microinjection, immersions, or with vapours using
various fixatives including aldehydes(glutaraldehyde),
osmium tetroxide,, tannic acid, or thiocarbohydrazide.
FIXATIVES- are chemicals that denature and
precipitate cellular macromolecules.

15. Some common fixatives:
1. GLUTARALDEHYDE- is a 5-carbon with an aldehyde
group at each end of the molecule. An aldehyde
groups reacts with amino groups and cross link with
the proteins into an insoluble network.
2. OSMIUM- heavy metal that reacts primarily with
fatty acids leading to the preservation of cellular
membranes.

16. TEM SAMPLE PREPARATION
 Rinsing of the specimen
After the fixation step, samples must be rinsed in
order to remove the excess fixative. To remove excess
glutaraldehyde from the samples, the specimen should
be subjected to a thorough but carefully conducted
rinsing procedure. Specimens can be washed in 0.1 M
cacodylic acid buffer (pH 7.3), starting with one time
for 10 min, and then three times for 20 min at 4 oC.

17. TEM SAMPLE PREPARATION
 Secondary fixation of the specimen
Specimen can be successfully stabilized for TEM
investigation by post fixation with 1% osmium
tetroxide prepared in 0.1 M cacodylic acid buffer (pH
7.3) for 1.5 hrs at room temperature (immersion
fixation).

18. TEM SAMPLE PREPARATION
 Dehydrating the specimen
For TEM investigation, specimen can be dehydrated in
a graded series of ethanol. More specifically, the
following protocol is useful: Dehydration of specimen
in 50% ethanol for 5 min, 70% ethanol for 10 min, 80%
ethanol for 10 min, 90% ethanol for 15 min, and 99.9%
ethanol (dried with a 4-mesh molecular sieve) twice
for 20 min at room temperature. This process allows
the water in the samples to be slowly exchanged
through liquids with lower surface tensions.

19. TEM SAMPLE PREPARATION
 Infiltration of the specimen with a transitional
solvent
The reason why this step is required is that the ethanol is
not miscible with the plastic embedding medium I found
most suitable for TEM investigation of the specimen
(mollicutes). The replacement of the dehydration solution
by another intermediary solvent (i.e., propylene oxide) is
thus necessary [1, 23, 26]. This process is essentially an
alcohol substitution. The immersion of mollicutes in
propylene oxide twice for 20 min at room temperature is
sufficient before attempting to embed the specimens in a
resin.

20. TEM SAMPLE PREPARATION
 Infiltration with resin and embedding the specimen
Mollicutes can be embedded in a variety of different media depending on the use (e.g.,
conventional TEM or immuno TEM). For conventional TEM of mollicutes, the epoxy
resin Durcupan ACM is quite suitable. The following protocol can be used: Immersion of
mollicutes in propylene-oxide/Durcupan-ACM (1:1; v/v) at room temperature overnight
(use gloves and a fume hood, and leave the specimen container open for the propylene
oxide to evaporate). The next day, the specimens should be immersed in a freshly
prepared Durcupan ACM mixture (pure) and left for 2 hrs at room temperature. A
second Durcupan ACM mixture (pure) is then prepared and used as the embedding
medium (free of air bubbles!).ible with the plastic embedding medium I found most
suitable for TEM investigation of mollicutes. The replacement of the dehydration
solution by another intermediary solvent (i.e., propylene oxide) is thus necessary [1, 23,
26]. This process is essentially an alcohol substitution. The immersion of mollicutes in
propylene oxide twice for 20 min at room temperature is sufficient before attempting to
embed the specimens in a resin. Polymerization of the epoxy mixture can be achieved by
placing the specimens in a drying cabinet for 2 days at 40 oC and for an additional 2 days
at 60 oC. Leaving the samples after heat polymerization for an additional 1-2 weeks at
room temperature can improve the subsequent cutting experience as the resin blocks
continue to harden during this time.

21. TEM SAMPLE PREPARATION
 Sectioning and staining of the specimen
The procedure for cutting specimens into semithin
and ultrathin slices (sections) is known as microtomy
and ultramicrotomy, respectively. Semithin sections
(0.5 μm to 2 μm) were typically stained with toluidine
blue for 1 min on a hot plate (70 oC to 90 oC),
examined by LM, and used for identifying the
specimen within the resin block before proceeding
with ultramicrotomy. Ultrathin sections (about 70 nm
to 90 nm) were typically stained with uranyl acetate
followed by lead citrate.

22. OTHER METHODS OF
SAMPLE PREPARATION
(TEM)

23. Cryofixation and the use of Frozen specimen
 CRYOFIXATION-like chemical fixatives it stops
metabolic processes and preserves biological
structure.
 The method involves ultra-rapid cooling of small
samples to liquid nitrogen temperature (-196 oC) or
below, thus stopping all motion and metabolic activity,
and preserving the internal structure by freezing all
fluid phases solid.

24.  The ultimate objective is to freeze the specimen so
rapidly (at 104 to 106 K per second) that ice crystals are
unable to form, or are prevented from growing
sufficiently large to cause damage to the specimen's
ultrastructure.

25. NEGATIVE STAINING
 The main purpose of negative-staining is to surround
or embed the biological object in a suitable electron
dense material which provides high contrast and good
preservation. This method is capable of providing
information about structural details often finer than
those visible in thin sections, replicas, or shadowed
specimens. In addition to the possibility of obtaining a
spectacular enhancement of contrast, negative-
staining has the advantage of speed and simplicity.

26.  The technique has mainly been used to examine
particulate (purified) specimens - e.g.. ribosomes,
enzyme molecules, viruses, bacteriophages,
microtubules, actin filaments, etc. at a resolution of
1.5-2.5 nm. This technique generally allows the shape,
size, and the surface structure of the object to be
studied as well as provide information about subunit
stoichiometries and symmetry in oligomeric
complexes. Any surface of the specimen accessible to
water can potentially be stained, and thus, that part of
the specimen will be imaged at high contrast.

27. SHADOW CASTING
 The grid containing the specimen are placed in sealed
chamber which is the evacuated by vacuum pump.
 The chamber contains a filament composed of a heavy
metal together with carbon.

29. The shadow cast by the nanoparticles (NPs) on metal deposition is
visible in the high-magnification images.

30. FREEZE FRACTURE REPLICATION
AND FREEZE ETCHING
 Small pieces of tissue are placed on a small metal disk
and rapidly frozen.
 The disk is then mounted on a cooled stage within a
vacuum chamber and a frozen tissue block is struck by
a knife edge.
 The resulting fracture plane spreads out from the point
of contact, splitting the tissue into two pieces.

31. Freeze fracturing can separate the two phospholipid leaflets that form
every cellular membrane
(a) A preparation of cells or tissues is
quickly frozen in liquid nitrogen at −196
°C, which instantly immobilizes cell
components. (b) The block of frozen cells
is fractured with a sharp blow from a cold
knife. The fracture plane is irregular, often
between the leaflets of the plasma or an
organelle membrane. (c) Membrane
proteins and particles remain bound to one
leaflet or the other, as illustrated in the
expanded view of a fractured membrane.

32.  shadowing
 coating specimen with a thin film of a heavy metal
 freeze-etching
 freeze specimen then fracture along lines of greatest
weakness (e.g., membranes)

34. GRID IS A SIEVE WOVEN FROM A THIN METAL WIRE,USUALLY
NICKEL OR COPPER GRIDS OF 3 mm DIAMETER ARE
COMMERCIALLY AVAILABLE WITH DIFFERENT MESH
SIZES(GENERALLY OF 100-200 µm SIZE)

35. Staphylococcus aureus platinum replica image shot on a TEM at
50,000X magnification

36. A TEM image of the polio virus.

37. CONCLUSION and
RECOMMENDATION
 Sample preparation for TEM involves more and some
different steps than those for SEM. Like in any multi-step
preparation procedure, virtually every step can affect the
quality of the final electron micrograph. A single mistake
in one of these steps will affect all remaining steps, and
thus the outcome of the entire study. It is therefore
important that the investigator plans and executes every
step in great detail. These procedures involve a significant
time commitment and require patience and skills that
come only with practice. It is important to mention that
most of the chemicals used in EM are dangerous.
Investigator must be aware of potential hazards such as
fire, chemical, electrical, and physical associated with these
items.

38. REFERENCES
 http://cms.mse.berkeley.edu/publications/MRS_Spring_20
04_Xu.pdf
 http://www.sciencepub.org/nature/0403/03-0171-
mahongbao-ns.pdf
 http://www.arl.army.mil/arlreports/2003/ARL-TR-3128.pdf
 http://stem.ornl.gov/papers/reviews/pdfs/NIST.pdf
 http://cimewww.epfl.ch/people/cayron/Fichiers/theseboo
k-chap3.pdf
 http://em-outreach.ucsd.edu/web-course/Sec-II.A.3/Sec-
II.A.3.html
 http://en.wikipedia.org/wiki/Cryofixation