electron microscopy

1. ELECTRON MICROSCOPY
 Dr. AMEENA THAHA
 23rd JULY 2020

2. HISTORY
 DERIVED FROM 2 GREEK WORDS
 MIKROS meaning small
 SKOPEO meaning look at
 Electron Microscope was invented by two Germans
 ERNST RUSKA & MAX KNOLL in 1931
 ERNST RUSKA –original model in 1933
 NOBEL PRIZE -1986

3. Uses a beam of accelerated electrons as a source of illumination
As the wavelength of electrons can be up to 1,00,000 times shorter
than that of visible light photons,
the electron microscope has a much better resolving power than a
light microscope.
It can reveal details of:
flagella
fimbriae
intra-cellular structures of cells

4. TYPES OF ELECTRON MICROSCOPE
• Transmission Electron Microscopy (TEM)
• Scanning Electron Microscopy (SEM)

5. Transmission Electron Microscopy
(TEM)
 Microscopy technique in which a beam of electrons is
transmitted through a specimen to form an image.
 Specimen - ultrathin section 20 -100 nm is viewed.
 Bacterial cells are thicker. Should be sliced into thin
layers

6. SPECIMEN PREPARATION
➢ Fixation - Glutaraldehyde or osmium tetroxide to stabilize cell structure.
➢ Dehydrated - Organic solvents (e.g., acetone or ethanol).*
➢ Embedding - Specimen is embedded in plastic polymer and is then hardened to form
a solid block.
➢ Slicing -Thin sections are cut from this block with a glass or diamond knife (
ultramicrotome)
➢ Staining
➢ Mounted on Metal Slide (Copper)

7. BASIC PRINCIPLES
 A heated tungsten filament in electron gun generates a beam of
electrons.
 Travels at high Speed  Bombarded on thin slice specimen.
 Electrons cannot pass through glass lens, therefore doughnut shaped
electromagnets called magnetic lenses are used.

8.  Column containing lenses and specimen is
under high vacuum to obtain a clear image.
 Specimen scatters some electrons pass
through are used to form an enlarged image 
on a fluorescent screen.
 A denser region in specimen scatters more
electrons darker image k/a Electron
dense.
 Electron-transparent regions - brighter.
 Image captured on photographic film as
permanent record.

9. Measures to increase contrast
 STAINING of specimen:
Increases contrast
Lead citrate and uranyl acetate
Heavy osmium atoms from osmium tetroxide fixative.
➢NEGATIVE STAINING
➢SHADOWING

10. NEGATIVE STAINING
• Specimen is spread out in a thin film
with heavy metals like phosphotungstic
acid or uranyl acetate.
• Heavy metals do not penetrate
specimen but render background dark
• Specimen appears bright.
• Excellent method to study structure of
viruses & bacterial gas vacuoles.
SHADOWING
• Specimen coated with a thin film
platinum or other heavy metal at 45
degree angle , so that the metal strikes the
micro-organism on only one side.
• Area coated with metal appears light in
photographs
• Uncoated side and the shadow region
created by the object appears dark.
• Useful in studying virus morphology,
prokaryotic flagella, and DNA.

11. Freeze-etching
 Cells are rapidly frozen in liquid
nitrogen & then warmed to -100°C
vacuum chamber.
 Knife precooled with liquid nitrogen
(-196°C) fractures the frozen cells
 The specimen left in high vacuum for
a minute or more.
 Ice sublimate away and uncover more
structural detail.
 Exposed surfaces are shadowed and
coated with layers of platinum and
carbon to form a replica of the surface.
 After the specimen removed
chemically replica studied in TEM
 3D view of intracellular structure.

13. TEM
Advantages
• Very powerful magnification and
resolution.
• Wide-range of applications - utilized in a
variety of different scientific, educational
and industrial fields
• Provide information on element and
compound structure .
• Images are high-quality and detailed.
Disadvantages
• Large and very expensive.
• Laborious sample preparation.
• Operation and analysis requires special
training.
• Samples are limited to those that are electron
transparent.
• Require special housing and maintenance.
• Images are black and white .

14. APPLICATION of TEM
 In medicine as a diagnostic tool – important in renal biopsies.
 Cellular tomography
– Used to obtaining detailed 3D structures of subcellular macromolecular objects.
 Cancer research – study of tumor cell ultrastructure .
 Toxicology – study the impacts of environmental pollution on the different
levels of biological organization.

15. SCANNING ELECTRON MICROSCOPE
 Produces an image from electrons released from
atoms on object’s surface.
 Used examine the surfaces of microorganisms in
great detail.
 Have resolution of 7 nm or less.
 Specimen preparation is relatively easy.
 Some cases , air-dried material can be examined
directly.

16. PRINCIPLE
 Incoming (primary) electrons
– can be “reflected” (backscattered) from bulk
specimen.
– can release secondary electrons.
 Primary electrons focused into small diameter electron
probe that scans across the specimen.
 Electrostatic or magnetic fields, applied at right angles
to the beam, used to change its direction of travel.
 By scanning simultaneously in two perpendicular
directions square or rectangular area of specimen (k/a
raster) covered.
 Image can be formed by collecting secondary electrons
from each point on the specimen.

17. SPECIMEN PREPRATION
 Sample coated with a thin layer of conductive material.
 Done using device called "sputter coater.”
 Sample placed in small chamber that is at a vacuum .
 Gold foil is placed in the instrument.
 Argon gas and electric field, electron to be removed from the argon  makes the
atoms positively charged.
 Argon ions gets attracted to negatively charged gold foil  knock gold atoms
from surface of gold foil.
 These gold atoms fall and settle onto the surface of the sample producing a thin
gold coating.

18. WORKING
 When the beam strikes a particular area,
surface atoms discharge  electrons called
secondary electrons trapped by a special
detector.
 Secondary electrons entering the detector
strike a scintillator emit light flashes 
photomultiplier converts to an electrical
current and amplifies.
 The signal sent to a cathode-ray tube
produces an image like television
picture  viewed or photographed.
 The number of secondary electrons
reaching the detector depends on the nature
of the specimen’s surface.

19.  Raised areas appear lighter on the
screen
 Depressions are darker.
 A realistic 3D image of the
microorganism’s surface results.
 Actual in situ location of
microorganisms in ecological
niches such as the human skin and
the lining of the gut also can be
examined.

20. SEM
Advantages
 Detailed 3D and topographical imaging
and the versatile information.
 Works very fast.
 Modern SEMs allow generation of data
in digital form.
 Most SEM samples require minimal
preparation actions.
.
DISADVANTAGES
• Expensive and large.
• Special training is required to operate.
• Preparation of samples can result in
artifacts.
• Limited to solid samples.
• Carry small risk of radiation exposure

21. APPLICATIONS of SEM
 Virology - for investigations of virus structure
 Cryo-electron microscopy – Images can be made of the surface of
frozen materials.
 3D tissue imaging
– Helps to know how cells are organized in a 3D network
– Their organization determines how cells can interact.
 Forensics - SEM reveals the presence of materials on evidences
that is otherwise undetectable
 SEM renders detailed 3-D images
– extremely small microorganisms
– anatomical pictures of insect, worm, spore, or other organic
Structures.

22. Comparison
TEM SEM
1. Transmitted electrons Scattered electrons
2. Electrons directly point towards sample Scattered electrons produce the image of sample after
microscope collects & counts the scattered electron
3. Seeks to see what is inside or beyond the surface Focuses on samples surface &its composition
4. Shows sample as a whole Shows sample bit by bit
5. Delivers a 2D picture Provides a 3D image
6. Has a 50 million magnification. Offers 2 million as max level of magnification
7. Resolution- 0.5 A° Resolution- 0.4m

23. SCANNING PROBE MICROSCOPY
 Most powerful new microscope
 Measure surface features by moving a sharp probe over the object’s
surface.
 The scanning tunneling microscope, invented in 1980.
 It can achieve magnifications of 100 million times
 Allow scientists to view atoms on the surface of a solid.
2 types:
1. Scanning Tunnelling Microscope
2. Atomic Force Microscope

24. SCANNING TUNNELING MICROSCOPE
 Gerd Binnig and Heinrich Rohrer invented in 1980.
 Won Nobel prize for physics with Ernst ruska.
 Needle like probe with point so sharp that often only one atom
at its tip.
 Probe lowered toward the specimen surface until its electron
cloud just touches the surface atoms.
 Small voltage is applied between the tip and specimen
 Electrons flow through a narrow channel in the electron
cloudsTunnelling current extraordinarily sensitive to
distance

25.  Arrangement of atoms on specimen surface
determined  moving probe tip back and
forth over the surface.
 Probe height constant above the specimen to
maintain a steady tunneling current.
 Its motion recorded and analyzed by a
computer  creates accurate 3D image of
the surface atoms.
 The surface map displayed on computer
screen or plotted on paper.
 The resolution is so great that individual
atoms are observed easily.

26.  Have major impact in biology.
 Used to directly view DNA and other biological molecules.
 Examine objects when they are immersed in water, used study
biological molecules(DNA).

27. ATOMIC FORCE MICROSCOPE
 Second type of scanning probe microscope.
 Moves a sharp probe over the specimen surface while
keeping the distance between the probe tip and the surface
constant.
 Exerting a very small amount of force on the tip enough
to maintain a constant distance but not enough force to
damage the surface.
 The vertical motion of the tip usually is followed by
measuring the deflection of a laser beam that strikes the
lever holding the probe.

28. Tip used to probe specimen
attached to cantilever.
As probe passes over surface
,cantilever deflected
vertically.
Laser beam directed towards
cantilever used to monitor
vertical movements.
Light reflected by cantilever
 detected by photodiode
 generates image.

29. USES
 Study surfaces that do not conduct
electricity well.
 Study interactions between the E. coli
GroES and GroEL chaperone proteins.
 To map plasmids by locating restriction
enzymes bound to specific sites.
 To follow behavior of living bacteria and
other cells.
 To visualize membrane proteins.

30. MICROSCOPE IMPORTANT FEATURES
Visible Light as source of illumination
1. Bright-field microscope Common,multi purpose, for live and preserved stained specimen.
Specimen -dark field-bright
2 Dark field microscope Best for observing live unstained specimens, specimen - bright field dark,
provides outline of specimen with reduced internal cellular details.
3. Phase contrast Used for live specimens, specimen is dark against bright background excellent
for internal cellular details.
UV light as source of illumination
1. Fluorescent microscope Specimen stained with fluorescent dye or combined with fluorescent
antibodies, emit visible light; specificity makes this microscope an excellent
diagnostic tool.
Electron beam forms image of specimen
1 TEM Section of specimen viewed under very high magnification ,finest detail
structure of cells and viruses shown, used only on preserved material.
2 SEM Scans and magnifies external surface of specimen ,produces striking 3D
images

31. References
 Prescott’s microbiology 8th edition.
 Bailey & scotts Diagnostic microbiology 14th edition.
 Manual of clinical Microbiology by Pfaller & James.H.Jorgensen 11th edition.