User will learn principle, instrumentation and applications of TEM. User will also know the difference between SEM and TEM

1. TRANSMISSION ELECTRON
MICROSCOPY
By: Dr. Sejal P. Gandhi
Dr. D. Y. Patil Institute of Pharmaceutical Sciences
and Research, Pimpri, Pune

2. Transmission electron microscopy (TEM) is a microscopy technique
where by 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 on to 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.
Principle of TEM

4. A transmission Electron Microscope is anologous to a slide projector as indicated by
Philips below

5. Working Concept
 TEM works much like a slide projector.
 A projector shines a beam of light through (transmits) the slide,
as the light passes through it is affected by the structures and
objects on the slide.
 These effects result in only certain parts of the light beam being
transmitted through certain parts of the slide.
 This transmitted beam is then projected onto the viewing screen,
forming an enlarged image of the slide.
 TEMs work the same way except that they shine a beam of
electrons (like the light) through the specimen (like the slide).
 Whatever part is transmitted is projected onto a phosphor screen
for the user to see.

6. INSTRUMENTATION

7. VACUUM

9.  The "Virtual Source" at the top represents the electron gun,
producing a stream of monochromatic electrons.
 This stream is focused to a small, thin, coherent beam by the use
of condenser lenses 1 and 2. The first lens (usually controlled by
the "spot size knob") largely determines the "spot size"; the
general size range of the final spot that strikes the sample.
 The second lens (usually controlled by the "intensity or
brightness knob" actually changes the size of the spot on the
sample; changing it from a wide dispersed spot to a pinpoint
beam.
 The beam is restricted by the condenser aperture (usually user
selectable), knocking out high angle electrons (those far from
the optic axis, the dotted line down the center)
 The beam strikes the specimen and parts of it are transmitted

10.  This transmitted portion is focused by the objective
lens into an image
 The image is passed down the column through the
projector lenses, being enlarged all the way.
 The image strikes the phosphor image screen and
light is generated, allowing the user to see the
image

11. Unscattered
electrons
Elastically
scattered
electrons
In - Elastically
scattered
electrons
Incident Electron Beam
Specimen
????

12. Source
Incident electrons which are transmitted through the thin
specimen without any interaction occurring inside the
specimen.
Utilization
The transmission of unscattered electrons is inversely
proportional to the specimen thickness.
Areas of the specimen that are thicker will have fewer
transmitted unscattered electrons and so will appear darker,
conversely the thinner areas will have more transmitted and thus
will appear lighter.
Unscattered Electrons

13. Unscattered
electrons
Elastically
scattered
electrons
In - Elastically
scattered
electrons
Incident Electron Beam
Specimen
????

14. Source
Incident electrons that are scattered (deflected from their
original path) by atoms in the specimen in an elastic fashion
(no loss of energy).
These scattered electrons are then transmitted through the
remaining portions of the specimen.
Utilization
•All electrons follow Bragg's Law and thus are scattered according
to Wavelength=2*Space between the atoms in the
specimen*sin(angle of scattering).
•All incident electrons have the same energy (thus wavelength) and
enter the specimen normal to its surface
Elasticity Scattered electrons
Use Bragg's law -  = 2d sin 

15. These "similar angle" scattered electrons can be collated
using magnetic lenses to form a pattern of spots; each
spot corresponding to a specific atomic spacing (a
plane).
This pattern can then yield information about the
orientation, atomic arrangements and phases
present in the area being examined.

16. Unscattered
electrons
Elastically
scattered
electrons
In - Elastically
scattered
electrons
Incident Electron Beam
Specimen
????

17. Source
Incident electrons that interact with specimen atoms in a
inelastic fashion, loosing energy during the interaction. These
electrons are then transmitted trough the rest of the specimen
Utilization
All electrons follow Bragg's Law and thus are scattered according
to Wavelength=2*Space between the atoms in the specimen*sin
(angle of scattering).
All incident electrons have the same energy(thus wavelength) and
enter the specimen normal to its surface
Inelastically Scattered Electrons

18. The inelastic loss of energy by
the incident electrons is
characteristic of the elements
that were interacted with.
These energies are unique to
each bonding state of each
element and thus can be used to
extract both compositional
and bonding (i.e. oxidation
state) information on the
specimen region being
examined.
Kakuchi Bands: Bands of
alternating light and dark lines
that are formed by inelastic
scattering interactions that are
related to atomic spacings in
the specimen.
These bands can be either
measured (their width is
inversely proportional to
atomic spacing) or "followed"
like a roadmap to the "real"
elasticity scattered electron
pattern.
Inelastically scattered electrons
Can be utilized two ways

19.  The acceleration voltage of up to date routine instruments is 120 to
200 kV.
 Medium-voltage instruments work at 200-500 kV to provide a
better transmission and resolution, and in
 high voltage electron microscopy (HVEM) the acceleration voltage
is in the range 500 kV to 3 MV.
 Acceleration voltage determines the velocity, wavelength and
hence the resolution (ability to distinguish the neighbouring
microstructural features) of the microscope.
Depending on the aim of the investigation and configuration of the
microscope, transmission electron microscopy can be categorized as:
Conventional Transmission Electron Microscopy
High Resolution Electron Microscopy
Analytical Electron Microscopy
Energy-Filtering Electron Microscopy
High Voltage Electron Microscopy
Dedicated Scanning Transmission Electron Microscopy

20. ULTRA THIN LAYER OF SPECIMEN

21. 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

22. • 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
Tissue preparation for TEM

23. Ultrathin
sections
on grid

24. mitochondria

25. SEM TEM
Type of electrons
Scattered, scanning
electrons
Transmitted electrons
High tension ~1 – 30 kV ~60 – 300 kV
Specimen thickness Any Typically <150 nm
Type of info 3D image of surface
2D projection image of inner
structure
Max. magnification Up to ~1 – 2 million times More than 50 million times
Max. FOV (Field of
view)
Large Limited
Optimal spatial
resolution
~0.5 nm < 50 pm
Image formation
Electrons are captured and
counted by detectors,
image on PC screen
Direct imaging on fluorescent
screen or PC screen with CCD
Operation Little or no sample
preparation, easy to use
Laborious sample preparation,
trained users required

27.  A Transmission Electron Microscope is ideal for a number of
different fields such as life sciences, nanotechnology, medical,
biological and material research, forensic analysis, gemology and
metallurgy as well as industry and education.
 TEMs provide topographical, morphological, compositional and
crystalline information.
 The images allow researchers to view samples on a molecular
level, making it possible to analyze structure and texture.
 This information is useful in the study of crystals and metals, but
also has industrial applications.
 TEMs can be used in semiconductor analysis and production and
the manufacturing of computer and silicon chips.
 Technology companies use TEMs to identify flaws, fractures and
damages to micro-sized objects; this data can help fix problems
and/or help to make a more durable, efficient product.
 Colleges and universities can utilize TEMs for research and
studies.
APPLICATIONS

28.  TEMs offer the most powerful magnification, potentially over
one million times or more
 TEMs have a wide-range of applications and can be utilized in
a variety of different scientific, educational and industrial fields
 TEMs provide information on element and compound structure
 Images are high-quality and detailed
 TEMs are able to yield information of surface features, shape,
size and structure
 They are easy to operate with proper training
ADVANTAGES

29. TEMs are large and very expensive
Laborious sample preparation
Potential artifacts from sample preparation
Operation and analysis requires special training
Samples are limited to those that are electron transparent, able
to tolerate the vacuum chamber and small enough to fit in the
chamber
TEMs require special housing and maintenance
Images are black and white
DISADVANTAGES