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THE
TRANSMISSION
ELECTRON
MICROSCOPE
(TEM)
By
Dr. S. PUGALENDHI, Emeritus
Professor
Dr. P. VIJAYAKUMARY,
Assistant Professor
Er. T. AYISHA NAZIBA, II-
M.Tech.(REE)
• Transmission electron microscopes (TEM) are microscopes that use a particle beam
of electrons to visualize specimens and generate a highly-magnified image. TEMs
can magnify objects up to 2 million times. In order to get a better idea of just how
small that is, think of how small a cell is. It is no wonder TEMs have become so
valuable within the biological and medical fields.
HOW DO TEMS WORK
• TEMs employ a high voltage electron beam in order to create an image.
• An electron gun at the top of a TEM emits electrons that travel through the
microscope’s vacuum tube. Rather than having a glass lens focusing the light (as in
the case of light microscopes), the TEM employs an electromagnetic lens which
focuses the electrons into a very fine beam.
• This beam then passes through the specimen, which is very thin, and the electrons
either scatter or hit a fluorescent screen at the bottom of the microscope.
• An image of the specimen with its assorted parts shown in different shades
according to its density appears on the screen. This image can be then studied
directly within the TEM or photographed. Figure 1 shows a diagram of a TEM and
its basic parts.
IMAGING
• The beam of electrons from the electron gun is focused into a small, thin, coherent beam by
the use of the condenser lens. This beam is restricted by the condenser aperture, which
excludes high angle electrons. The beam then strikes the specimen and parts of it are
transmitted depending upon the thickness and electron transparency of the specimen.
• This transmitted portion is focused by the objective lens into an image on phosphor screen
or charge coupled device (CCD) camera.
• Optional objective apertures can be used to enhance the contrast by blocking out high-
angle diffracted electrons. The image then passed down the column through the
intermediate and projector lenses, is enlarged all the way.
• The image strikes the phosphor screen and light is generated, allowing the user to see the
image. The darker areas of the image represent those areas of the sample that fewer
electrons are transmitted through while the lighter areas of the image represent those areas
of the sample that more electrons were transmitted through.
DIFFRACTION
• As the electrons pass through the sample, they are scattered by the electrostatic
potential set up by the constituent elements in the specimen.
• After passing through the specimen they pass through the electromagnetic
objective lens which focuses all the electrons scattered from one point of the
specimen into one point in the image plane.
• Also, shown in fig 2 is a dotted line where the electrons scattered in the same
direction by the sample are collected into a single point.
• This is the back focal plane of the objective lens and is where the diffraction pattern
is formed.
DIFFERENCES BETWEEN A TEM AND
A LIGHT MICROSCOPE
• Although TEMs and light microscopes operate on the same basic principles, there
are several differences between the two.
• The main difference is that TEMs use electrons rather than light in order to magnify
images.
• The power of the light microscope is limited by the wavelength of light and can
magnify something up to 2,000 times. Electron microscopes, on the other hand, can
produce much more highly magnified images because the beam of electrons has a
smaller wavelength which creates images of higher resolution. (Resolution is the
degree of sharpness of an image.)
Cotton stem; area in the circle is the
phloem tissue. Light microscope x250
Enlarged image of cotton phloem tissue
showing a sieve element (top cell) and a
companion cell (bottom cell), TEM x8,000.
TEM SPECIMENS PREPARATION
• Specimens must be very thin so that electrons are able to pass through the tissue.
• This may be done by cutting very thin slices of a specimen’s tissue using an ultramicrotome.
• The tissue must first be put in a chemical solution to preserve the cell structure.
• The tissue must also be completely dehydrated (all water removed).
• Once preserved and dehydrated, tissue samples are placed in hard, clean plastic.
• The plastic supports the tissue while it is being thinly cut with the ultramicrotome. After
sections are cut and mounted on grids, (tiny circular disks with openings,) a solution of lead
is used to stain the tissue.
• The lead provides contrast to the tissue by staining certain cell parts. When placed in the
electron microscope, the electrons are scattered by the lead. They do not penetrate the
tissue or hit the fluorescent screen, leaving those areas dark.
ULTRAMICROTOME & MICROTOME
GRID
10. The Transmission Electron Microscope (TEM).pptx

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10. The Transmission Electron Microscope (TEM).pptx

  • 1. THE TRANSMISSION ELECTRON MICROSCOPE (TEM) By Dr. S. PUGALENDHI, Emeritus Professor Dr. P. VIJAYAKUMARY, Assistant Professor Er. T. AYISHA NAZIBA, II- M.Tech.(REE)
  • 2. • Transmission electron microscopes (TEM) are microscopes that use a particle beam of electrons to visualize specimens and generate a highly-magnified image. TEMs can magnify objects up to 2 million times. In order to get a better idea of just how small that is, think of how small a cell is. It is no wonder TEMs have become so valuable within the biological and medical fields.
  • 3. HOW DO TEMS WORK • TEMs employ a high voltage electron beam in order to create an image. • An electron gun at the top of a TEM emits electrons that travel through the microscope’s vacuum tube. Rather than having a glass lens focusing the light (as in the case of light microscopes), the TEM employs an electromagnetic lens which focuses the electrons into a very fine beam. • This beam then passes through the specimen, which is very thin, and the electrons either scatter or hit a fluorescent screen at the bottom of the microscope. • An image of the specimen with its assorted parts shown in different shades according to its density appears on the screen. This image can be then studied directly within the TEM or photographed. Figure 1 shows a diagram of a TEM and its basic parts.
  • 4.
  • 5. IMAGING • The beam of electrons from the electron gun is focused into a small, thin, coherent beam by the use of the condenser lens. This beam is restricted by the condenser aperture, which excludes high angle electrons. The beam then strikes the specimen and parts of it are transmitted depending upon the thickness and electron transparency of the specimen. • This transmitted portion is focused by the objective lens into an image on phosphor screen or charge coupled device (CCD) camera. • Optional objective apertures can be used to enhance the contrast by blocking out high- angle diffracted electrons. The image then passed down the column through the intermediate and projector lenses, is enlarged all the way. • The image strikes the phosphor screen and light is generated, allowing the user to see the image. The darker areas of the image represent those areas of the sample that fewer electrons are transmitted through while the lighter areas of the image represent those areas of the sample that more electrons were transmitted through.
  • 6. DIFFRACTION • As the electrons pass through the sample, they are scattered by the electrostatic potential set up by the constituent elements in the specimen. • After passing through the specimen they pass through the electromagnetic objective lens which focuses all the electrons scattered from one point of the specimen into one point in the image plane. • Also, shown in fig 2 is a dotted line where the electrons scattered in the same direction by the sample are collected into a single point. • This is the back focal plane of the objective lens and is where the diffraction pattern is formed.
  • 7. DIFFERENCES BETWEEN A TEM AND A LIGHT MICROSCOPE • Although TEMs and light microscopes operate on the same basic principles, there are several differences between the two. • The main difference is that TEMs use electrons rather than light in order to magnify images. • The power of the light microscope is limited by the wavelength of light and can magnify something up to 2,000 times. Electron microscopes, on the other hand, can produce much more highly magnified images because the beam of electrons has a smaller wavelength which creates images of higher resolution. (Resolution is the degree of sharpness of an image.)
  • 8. Cotton stem; area in the circle is the phloem tissue. Light microscope x250 Enlarged image of cotton phloem tissue showing a sieve element (top cell) and a companion cell (bottom cell), TEM x8,000.
  • 9. TEM SPECIMENS PREPARATION • Specimens must be very thin so that electrons are able to pass through the tissue. • This may be done by cutting very thin slices of a specimen’s tissue using an ultramicrotome. • The tissue must first be put in a chemical solution to preserve the cell structure. • The tissue must also be completely dehydrated (all water removed). • Once preserved and dehydrated, tissue samples are placed in hard, clean plastic. • The plastic supports the tissue while it is being thinly cut with the ultramicrotome. After sections are cut and mounted on grids, (tiny circular disks with openings,) a solution of lead is used to stain the tissue. • The lead provides contrast to the tissue by staining certain cell parts. When placed in the electron microscope, the electrons are scattered by the lead. They do not penetrate the tissue or hit the fluorescent screen, leaving those areas dark.