Electron microscopy is a technique that uses beams of electrons instead of light to view objects. There are two main types: transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM uses electrons transmitted through an ultra-thin sample to form magnified images, allowing visualization of structures as small as single atoms. SEM scans a focused beam of electrons across a sample to produce high-resolution 3D images of surface topology and composition. Newer techniques like scanning tunneling microscopy can achieve even higher resolution down to fractions of a nanometer. Electron microscopy has enabled significant advances in fields like materials science, biology, and nanotechnology.

1. Electron Microscopy
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Pt nanoparticles

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 nanometers, which are formed
by controlled use of electrons in vacuum
captured on a phosphorescent screen

12. Why were the EMs ad vented?
• 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. 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 visualization of molecules and
individual atoms, also in motion”

17. 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.

18. 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.

19. 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.

20. 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.

21. • By taking multiple images of a
single TEM sample at differing
angles,
• typically in 1° increments,
• 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.
Gold particles on E. coli appear
as bright white dots due to the
higher percentage of
backscattered electrons
compared to the low atomic
weight elements in the
specimen

22. Scanning TEM (STEM)
• Modified type of TEM
• by the addition of a system that raster 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

23. 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.

24. 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.

25. 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

26. Salient features
• Electrons are used to create images of the surface of
specimen - topology
• Resolution of objects of nearly 1 nm
• Magnification up to 500000 x (250 times > light
microscopes)
• 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

27. 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

28. 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”
• Visualization of intra cellular changes

29. 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

30. References:-
• 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
• www.Wikipedia.en.us/electron microscopy
• www.slideshare.com