1. Electron Microscopy

2. The Central player - (e)
• The electron “e” is an elementary particle
• Also called corpuscle
• carries a negative charge.
• the electron was discovered by J. J.
Thompson in 1897
• e is a constituent of the atom
• 1000 times smaller than a hydrogen atom.
• the mass of the electron 1/1836 of that of a
proton.

3. The milestones

4. The Wave Properties
• In 1924, the wave-particle dualism was
postulated by de Broglie (Nobel Prize 1929).
• All moving matter has wave properties with
the wavelength λ being inversely related to
the momentum p by
λ = h / p = h / mv
(h : Planck constant; m : mass; v : velocity)

5. The Wavelength
• Resolving power of EM is from Wave properties of
electrons
• Limit of resolution is indirectly proportional to the
wavelength of the illuminating light
• ie, longer the wavelength, lesser is the resolution
• λ = √150 / V, where
• λ – wavelength in Angstroms, V – accelerating
voltage in volts

6. The electron wave
• The generation of a monochromatic and
coherent electron beam is important
• Design of modern electron microscopes is
based on this concept

7. Scheme of electron-matter
interactions arising
from the impact of an electron beam
onto a specimen.
A signal below the specimen is
observable if the
thickness is small enough to allow
some electrons to
pass through

8. Elastic Electron Interactions
• no energy is transferred from the electron to
the sample.
• These signals are mainly exploited in
- Transmission Electron Microscopy and
- Electron diffraction methods.

9. Inelastic Electron Interactions
- Energy is transferred from the electrons to the
specimen
- The energy transferred can cause different
signals such as
- X-rays,
- Auger electrons
- secondary electrons,
- plasmons,
- phonons,
- UV quanta or cathodoluminescence.
• Used in Analytical Electron Microscopy … SEM

10. What is Electron Microscopy?
• Electron microscopy is a diagnostic tool with
diversified combination of techniques ……
• that offer unique possibilities to gain insights
into
- structure,
- topology,
- morphology, and
- composition of a material.

11. What is an Electron Microscope ?
• A special type of microscope having a high
resolution of images, able to magnify
objects in nanometres, which are formed
by controlled use of electrons in vacuum
captured on a phosphorescent screen

12. Why were the EMs advented?
• To study objects of < 0.2 micrometer
• For analysis of sub cellular structures
• Intra cellular pathogens - viruses
• Cell metabolism
• Study of minute structures in the
nature
Greater resolving power of the EMs than
light microscope
• An EM can magnify structures from
100 – 250000 times than light
microscopy

14. The novelty of EMs from others
• Beam of Electrons …… instead of a beam of light
• Electro-magnetic lens ………..instead of Ground glass
lenses
• Cylindrical Vacuum column - Electrons should travel
in vacuum to avoid collisions with air molecules that
cause scattering of electrons distorting the image

15. Comparison of lens system of Light & Electron
Microscope

16. TYPES OF Electron Microscopy
• Transmission Electron Microscopy (TEM)
- Bright Field (BF)/ Dark Field (DF)
- High-Resolution Transmission Electron Microscopy
(HRTEM)
- Energy Filtered Transmission Electron Microscopy
(EFTEM)
- Electron Diffraction (ED)
• Scanning Transmission Electron Microscopy (STEM)
- Bright Field (BF)/ Dark Field (DF)
- High-Angle Annular Dark Field (HAADF-STEM)

17. TYPES OF Electron Microscopy
Analytical Electron Microscopy (AEM)
- X-ray spectroscopy
- Electron Energy Loss Spectroscopy (EELS)
- Electron Spectroscopic Imaging (ESI)
Scanning Electron Microscopy (SEM)
- Secondary Electron Imaging (SE)
- Back-scattered Electron Imaging (BSE)

18. Commonly used EMs in biology
• Transmission Electron Microscope
• Scanning Electron Microscope
“ mainly for various life forms and microbes”
• Scanning tunneling microscope
• Atomic Force Microscope
“ Actual visualisation of molecules and
individual atoms, also in motion”

19. Transmission Electron
Microscopy
The first TEM was built by Max Knoll and Ernst Ruska in 1931, with
this group developing the first TEM with resolving power greater
than that of light in 1933 and the first commercial TEM in 1939.

20. TEM - Definition
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.

21. Applications
• TEMs are capable of imaging at a significantly higher
resolution than light microscopes, owing to the small de Broglie
wavelength of electrons.
• to examine fine detail—even as small as a single
column of atoms, which is tens of thousands times smaller than the smallest
resolvable object in a light microscope.
• application in Biological sciences like cancer research,
virology, materials science as well as pollution,
nanotechnology, and semiconductor research.
• Application in chemical & physical sciences like in
chemical identity, crystal orientation, electronic
structure and sample induced electron phase shift as
well as the regular absorption based imaging.

22. - vacuum system in which the electrons
travel,
- an electron emission source for generation
The TEM components of the electron stream (tungsten filament,
or a lanthanum hexaboride (LaB6)),
- Voltage source 100 – 300 kV
- a series of electromagnetic lenses, and
electrostatic plates.
- The latter two allow the operator to guide
and manipulate the beam as required.
- Also required is an Insertion device to allow
the insertion into, motion within, and
removal of specimens from the beam path.
- Imaging devices are subsequently used to
create an image from the electrons that exit
the system on Phosphor screen having Zinc
sulphide

23. Different types of TEM
• Bright Field (BF) - BFTEM
• Dark Field (DF) - DFTEM
• High-Resolution Transmission Electron
Microscopy (HRTEM)
• Energy Filtered Transmission Electron
Microscopy (EFTEM)
• Electron Diffraction (ED)

24. • The most common mode of operation for a
TEM is the bright field imaging mode.
• concept of “mass-thickness contrast”
Bright Field Imaging is
A TEM image of the polio virus. The
polio virus is 30 nm in size.
• “As the thickness of the specimen
increases, the contrast also increases”
• Thicker regions of the sample, or regions
with a higher atomic number will appear
dark,
• regions with no sample in the beam path
will appear bright – hence the term "bright
field".
• The image is in effect assumed to be a
simple two dimensional projection of the
sample
(modelled via Beer's law)

25. • Electrons passed through Crystal
Diffraction Contrast with equidistant lattice planes
Imaging • With regular spacing between
Crystalline diffraction pattern from a the scattering centers ,
twinned grain of FCC Austenitic steel
• coherent constructive
interference of the scattered
electron in certain directions
happens
• and thereby three-dimensional
secondary wavelets or
diffracted beams are generated
• This phenomenon is called Bragg
diffraction.
• These beams are captured on
image screen

26. Electron Energy Loss Spectroscopy
• electrons can be rejected based upon the
voltage using magnetic sector based devices
known as EELS spectrometers.
• These devices allow for the selection of particular energy values,
• EELS spectrometers can be operated in both
spectroscopic and imaging modes,
• allowing for isolation or rejection of elastically
scattered beams.
• EELS imaging can be used to enhance contrast
in observed images, including both bright field
and diffraction.

27. High resolution TEM - HRTEM
• Crystal structure can also be investigated by high-
resolution transmission electron microscopy (HRTEM),
• HRTEM is also known as phase contrast.
• In a specimen of uniform thickness, the images are
formed due to differences in phase of electron waves,
which is caused by specimen interaction.
• Image formation is given by the complex modulus of
the incoming electron beams.
• The image is dependent on the number of electrons
hitting the screen,
• it can be manipulated to provide more information
about the sample as in complex phase retrieval
techniques.

28. • By taking multiple images of a
single TEM sample at differing
3D Imaging angles,
Three dimensional imaging -A TEM • typically in 1° increments,
image of a parapoxavirus
• a set of images known as a "tilt
series" can be collected.
• This methodology was proposed
in the 1970s by Walter Hoppe.
• Under absorption contrast
conditions, this set of images
can be used to construct a
three-dimensional
representation of the sample.[36]

29. Scanning TEM (STEM)
• Modified type of TEM
•
• by the addition of a system that rasters the beam
across the sample to form the image, combined with
suitable detectors.
• The STEM uses magnetic lenses to focus a beam of
electrons
• The image is formed not by secondary electrons as in
SEM but by primary electrons coming through the
specimen

30. Scanning Transmission Electron Microscopy

31. • HAADF- STEM
• Also called “Z- contrast imaging”
High angular annular dark
field – STEM
• In this method small clusters, single
atoms of heavy atoms (e.g. in catalysts)
Comparison of TEM & STEM can be recognized in a matrix of light
(a) TEM and (b) HAADF-STEM image
of atoms
Pd balls on silica. • Because the contrast produced is high
The overlap regions are relatively
dark in (a) and bright in (b),
respectively.
• Eg: In the TEM image, the
overlapping of specimen leads to a
darker contrast
whereas
in the HAADF-STEM image the
contrast in the intersection
becomes brighter than in the
specimen.

32. Cryoelectron microscopy
• Cryoelectron Tomography (cryo-ET)
• Nano sized intra cellular structures are studied
• In unfixed , unstained, hydrated , flash frozen cells
• It bridges the cellular & molecular research
• This technique facilitate visualisation of molecular
structure of proteins and large molecules.
• Cryoelectron microscopy involves vitrification of the
macromolecular assemblies
• 3D images can also be generated

33. Limitations of TEM
• Many materials require extensive sample preparation
• Difficult to produce a very thin sample
• relatively time consuming process with a low throughput of
samples.
• The structure of the sample may change during the
preparation process.
• Small field of view may not give conclusive result of the
whole sample.

34. Biological Sample preparation
• Sample thickness – less than 1 mm3
• Rapid fixation with least cell damage –
Gluteraldehyde and Osmium tetroxide
- Gluteraldehyde has aldehyde groups which bonds with
amino groups of proteins, forming insoluble complexes
- OsO4 binds to cell membranes containing fatty acids
• Dehydration – alcohol series
• Embedding – Epoxy resins (Epon or araldite)
• Section – 0.1 micrometre using ultramicrotome
(Ideal – 70-90 nm thickness)
Staining – Uranyl acetate , Lead citrate

35. Cryofixation
• By rapid freezing – ultra freezing methods
“ Done to avoid ice crystal formation which damage the
fragile intra cellular str.”
• Water becomes frozen in liquid state , so that ice is
not formed -- VITRIFICATION
• Liquid propane (-42 deg C)
• Liquid Helium (-273 deg C)
• High pressure freezing with jets of Liq N
• Adv: Less time consuming, enzymology possible

36. Different techniques for TEM
• Negative staining
• Shadow casting
• Freeze fracture replication
• Freeze etching

37. • Heavy metal deposits are
collected onto specimen grid,
1. Negative Staining tq
Human papilloma virus by negative
except where the particle is
staining present
• The specimen appears bright
on the view screen

38. • Viruses, DNA, RNA
visualisation
2. Shadow Casting tq • The grids with specimen is
placed in a sealed chamber
• Vacuum is created
• Heated platinum+carbon
filament deposits metal onto
the surface directly in line ,
creating shadow
• Areas of shadow appear
bright on view screen
• The image in photographs
are reversed – objects appear
bright with a dark shadow

39. 3. Freeze Fracturing Tq
Step 3
Step 4
Step 5

40. Definition of SEM
• An electron microscope that produces images
of a sample by scanning over it with a focused
beam of electrons.
• The incident electrons interact with electrons
in the sample, producing various signals that
can be detected and
• contain information about the sample's
surface topography and composition.

41. The electron beams
• The types of signals produced by a SEM
include
- secondary electrons,
- back-scattered electrons (BSE),
- X-rays,
- light rays (cathodoluminescence),
- A standard SEM uses Secondary electrons &
Back scattered electrons

42. Salient features
• Electrons are used to create images of the surface of
specimen - topology
• Resolution of objects of nearly 1 nm
• Magnification upto 500000 x (250 times > light
microcopes)
• secondary electrons (SE), backscattered electrons
(BSE) are utilized for imaging
• specimens can be observed in high vacuum, low
vacuum and
• In Environmental SEM specimens can be observed in
wet condition.
• Gives 3D views of the exteriors of the objects like
cells, microbes or surfaces

43. 3D data measurement
• 3D data can be measured in the SEM with
different methods such as:
- photogrammetry (2 or 3 images from tilted
specimen)
- photometric stereo (use of 4 images from BSE
detector)
- inverse reconstruction using electron-material
interactive models[31][32]
• Possible applications are roughness measurement, measurement of fractal
dimension, corrosion measurement and height step measurement.

44. Biological sample preparation
• Chemical fixation with Gluteraldehyde, optionally with OsO4 – for soft tissues
• No fixation needed for dry specimen like bones, feathers etc
• Dehydration by replacement of water in the cells with organic solvents such as
ethanol or acetone, and replacement of these solvents in turn with a
transitional fluid such as liquid carbon dioxide by critical point drying.
• The carbon dioxide is removed while in a supercritical state, so that no gas-
liquid interface is formed within the sample during drying.
• The dry specimen is mounted on a specimen stub using epoxy resin
• ultrathin coating done by low-vacuum sputter coating or by high-vacuum
evaporation.
• Conductive materials in current use for specimen coating include gold,
gold/palladium alloy, platinum, osmium,[12] iridium, tungsten, chromium, and
graphite.

45. SEM - Cryo-imaging
• SEM is equipped with a cold stage for cryo-microscopy,
• Cryofixation is used and low-temperature scanning
electron microscopy performed on the cryogenically
fixed specimens.
- Cryo-fixed specimens are cryo-fractured under vacuum in a
special apparatus to reveal internal structure,
- sputter-coated, and transferred onto the SEM cryo-stage
while still frozen.
• Low-temperature scanning electron microscopy is also
applicable to the imaging of temperature-sensitive
materials such as ice and fats.
• Freeze-fracturing, freeze-etch or freeze-and-break is a
preparation method particularly useful for examining
lipid membranes and their incorporated proteins

46. SEM - Cathodolumiscence
• Cathodoluminescence means the “ emission of light
occurs when atoms are excited by high-energy electrons”
• It is analogous to UV-induced fluorescence,
Eg: Cathodoluminescence is most commonly experienced in cathode ray
tube in television sets and computer CRT monitors.
• In the SEM, CL detectors display an emission spectrum or
an image of the distribution of cathodoluminescence
emitted by the specimen “ in real colour”
• very powerful probe for
studying nanoscale features and defects.

47. SEM - X-ray microanalysis
• X-rays, which are produced by the interaction of
electrons with the sample,
• may be detected in an SEM equipped for
- energy-dispersive X-ray spectroscopy (EDXS) or
- wavelength dispersive X-ray spectroscopy
(WDXS).

48. Environmental SEM ( ESEM)
• Environmental SEM (ESEM) in the late 1980s
• samples are observed in low-pressure gaseous environments and
high relative humidity (up to 100%).
• ESEM is especially useful for non-metallic and biological materials
- because coating with carbon or gold is unnecessary.
- Uncoated Plastics and Elastomers & uncoated biological samples.
- ESEM makes it possible to perform X-ray microanalysis on
uncoated non-conductive specimens.
- ESEM may be the preferred tool for electron microscopy of unique
samples from criminal or civil actions, where “forensic
analysis” may need to be repeated by several different experts.

49. Scanning tunneling microscopy
• 1980 – Gerd Bennig and Heinrich Rohrer invented STM
• Also called Scanning Probe microscopes
• Object resolution 0.1-0.01 nm
• Thin wire probe made of platinum - iridium is used to trace the
surface of the object
• Electrons from the probe overlap with electron from the surface
- tunnel into one another’s clouds
- tunnels create a current as the probe moves on the uneven surface of the
specimen

50. The applications
• The STM can be used in ultra high vacuum, air, water, and
various other liquid or gaseous environments
• and at temperatures ranging from near zero to a few
hundred degrees Celsius
• First movie made using STM – “individual fibrin molecule
forming a clot”
• Live specimen examination – as in “Virus infected cells
exploding and releasing new viruses”
• Visualisation of intra cellular changes

51. Atomic force microscope
• Advanced type of EM
• Three dimensional imaging
• Measurement of structures at the level of an atom
• To study DNA, especially the base pairs by
detecting differences in density
• Used under water to study chemical reactions at
living cell surfaces
• Cell wall chemical composition visualisation
• Used to measure forces – as in protein unfolding,
polysaccharide flexibility etc

52. Low Voltage Electron Microscope- LVEM
• The low-voltage electron microscope (LVEM) is a
combination of SEM, TEM and STEM in one instrument
• operated at relatively low electron accelerating voltage
of 5 kV.
• Low voltage increases image contrast which is especially
important for biological specimens.
• This increase in contrast eliminates the need to stain.
• Sectioned samples need to be thinner than they would
be for conventional TEM (20–65 nm).

53. Recent advances
The effect of metallic nanoparticles
on cells was probed by treating two
cell lines with C-coated Cu
nanoparticles. The up-take of these
nanoparticles was shown by HAADF-
STEM that reveal them as bright
patches inside the cells (s. image).
Nanoparticle Cytotoxicity Depends
on Intracellular Solubility:
Comparison of Stabilized Copper
Metal and Degradable Copper Oxide
Nanoparticles
A. M. Studer, L. K. Limbach, L. Van
Duc, F. Krumeich, E. K. Athanassiou, L.
C. Gerber, H. Moch, and W. J. Stark
Toxicology Lett. 197 (2010) 169-174
DOI

54. Inner Shell Ionisation – X-ray or Auger electrons
• When an electron drops down from a higher
level to fill the vacancy in an atom.
• By this process, the atom can relax but the
excess energy has to be given away.
• This excess energy of the electron, causes
difference between the energy levels.
• The process of getting rid of the additional
energy generates either of a characteristic X-
ray or of an Auger electron.