slides include basic discussion on electrom microscopy and specially transmission electron microscopy. hope this will give you all the requires info.

1. INTRODUCTION AND HISTORY
• Electron microscopes were developed due to the limitations of light microscopes
which are limited by the physics of light
• In the early 1930’s this theoretical limit had been reached and there was a scientific
desire to see the fine details of the interior structures of organic cells (nucleus,
mitochondria etc)
• This required 10,000x plus magnification which was not possible using current
optical microscopes
• The first electromagnetic lens was developed in 1926 by Hans Busch.
• The physicist Ernst Ruska and the electrical engineer Max Knoll constructed the
prototype electron microscope in 1931.
• The transmission electron microscope was the first type of electron microscope to
be developed and is pattern exactly on the light transmission microscope except
that a focused beam of electrons is used instead of light” to see through” the
specimen.
• The first scanning electron microscope debuted in 1938 (Von ) with the first
commercial instruments around 1965.

2. Electron microscope
• A special type of microscope having a high resolution images, able to ,magnify objects in
nanometer, which are formed by controlled use of electrons in vaccum captured on a
phosphorescent screen.
Components of electron microscope
1.Electron gun- electron beam is generated in the electron gun. Two basic types of guns are
used:
~ Thermionic gun- it is based on the two types of filaments tungsten and Lanthanum
Hexaboride.
~Field emission gun- it employs either a thermally assisted cold field emitter.
2.Condensor lens- it collects the light rays and focuses them on the object it produces
illumination that is uniformly bright.
3.Objective lens- it is a strong lens and has highly concentrated magnetic field and short focal
length.
4.Fluorescent screen-A tool to visualize TEM images and diffraction patterns. A phosphor on a
fluorescent screen is excited through electron collision . It is composed of a matrix of zinc
sulfide and aluminum , etc .
5. Vaccum system –the electron beam must be generated and used in a high vaccum so that
the electrons are not deflected by gas molecules and the filament and specimen are not
contaminated .

3. LIGHT MICROSCOPE AND ELECTRON
MICROSCOPE
LIGHT
• BULB
• LENS MADE BY GLASS
• SPECIMEN STAGE MADE BY METAL
GRID,GLASS SLIDE USED
• SPECIMEN MOUNTED IN AIR
• MAGNIFICATION UPTO 1000
• RESOLVING POWER IS
0.4(MICROMETER)
ELECTRON
• ELECTRON GUN
• ELECTROMAGNETIC CONDENSOR
LENS
• METAL GRID
• SPECIMEN
• MOUNTED IN VACCUM
• MAGNIFICATION UPTO 2LAKH
• RESOLVING POWER IS 100TIMES
MORE THAN LIGHT

4. TRANSMISSION ELECTRON
MICROSCOPY
• TEM is a unique tool in characterization of materials crystal structures and
microstructures simultaneously by diffraction and imaging techniques and form images.
• PRINCIPLE
• Electrons are made to pass through the specimen and the image is formed on the
fluorescent screen, either by using the transmitted beam or by using the diffracted
beam.
COMPONENTS AND WORKING
• Tungsten filament generates a beam of electrons that is then focused on the specimen
by the condenser
• Magnetic lenses are used to focus the beam.
• The column containing the lenses and specimen must be under high vaccum to obtain a
clear image because electrons are diffracted by collisions with air molecules
• Magnetic lenses form the enlarged image visible of the specimen on a fluorescent
screen
• Photographic film –the screen can also be moved aside and the image captured on
photographic film as a permanent record.

5. Working:
~Stream of electrons are produced by the electron gun and is made to fall over
the specimen using the magnetic condensing lens.
~ Based on the angle of incidence the beam is partially transmitted and partially
diffracted. Both these beams are recombined at the E-wald sphere to form the image
~The combined image is called the phase contrast image.
~In order to increase the intensity and the contrast of the image, an amplitude
contrast has to be obtained. This can be achieved only by using the transmitting beam
and thus the diffracted beam can be eliminated.
~ Now in order to eliminate the diffracted beam, the resultant beam is passed
through the magnetic objective lens and the aperture. The aperture is adjusted in such
a way that the diffracted image is eliminated. Thus, the final image obtained due to
transmitted beam alone is passed through the projector lens for further magnification.
~The magnified image is recorded in fluorescent screen or CCD. This high
contrast image is called Bright Field Image.
~ Also, it has to be noted that the bright field image obtained is purely due to the
elastic scattering (no energy change) i.e., due to transmitted beam alone

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

8. SAMPLE PREPARATION
.
This includes: fixation, rinsing, post fixation, dehydration and infiltration.
1) Fixation-This is done to preserve the sample and to prevent further deterioration so that it appears as close as
possible to the living state, although it is dead now. It stabilizes the cell structure. There is minimum alteration to cell
morphology and volume. Glutaraldehyde is often used as the fixative in TEM. As a result of glutaraldehyde fixation the
protein molecules are covalently cross linked to their neighbors.
2) Rinsing-The samples should be washed with a buffer to maintain the pH. For this purpose, sodium cacodylate buffer
is often used which has an effective buffering range of 5.1-7.4. The sodium cacodylate buffer thus prevents excess
acidity which may result from tissue fixation during microscopy.
3) Post fixation-A secondary fixation with osmium tetroxide (OsO4), which is to increase the stability and
contrast of fine structure. OsO4 binds phospholipid head regions, which creating contrast with the neighbouring
protoplasm (cytoplasm). OsO4 helps in the stabilization of many proteins by transforming them into gels without
destroying the structural features. Tissue proteins, which are stabilized by OsO4 and does not coagulated b alcohol
during dehydration.
For imaging electrons scatterring ,heavy metals like uranium and lead are used and thus give contrast between
different structures. Thus we add more electron density to the internal structures.
4) Dehydration-The water content in the tissue sample should be replaced with an organic solvent since the epoxy resin
used in infiltration and embedding step are not miscible with water.
5) Infiltration-Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the space to give hard plastic
material which will tolerate the pressure of cutting.
6) Embedding:-After processing the next step is embedding. This is done using flat molds.
7) Polymerization-Next is polymerization step in which the resin is allowed to set overnight at a temperature of 60
degree in an oven.
8) Sectioning-The specimen must be cut into very thin sections for electron microscopy so that the electrons are
semitransparent to electrons. These sections are cut on an ultramicrotome which is a device with a glass or diamond
knife. For best resolution the sections must be 30 to 60 nm. The resin block can be made ready for the sectioning by
trimming it at the tip with a razor blade or black trimmer so that the smallest cutting face is available. Fix the block to a
microtome and cut the sections. Sections float onto a surface of liquid held in trough and remain together in a form of
ribbon. Freshly distilled water is generally used to fill the trough. These sections are then collected onto a copper grid
and viewed under the microscope.

10. APPLICATIONS,ADVANTAGES AND DISADVANTAGES OF
TEM
• TEM APPLICATIONS
• 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.
Advantages
• A Transmission Electron Microscope is an impressive instrument with a number of advantages such as:
• 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
• Disadvantages
• TEMs are large and very expensive
• Laborious sample preparation
• Potential artifacts from sample preparation

13. Fig.saccharomyces under tem