Microscopy: Transmission Electron Microscope (TEM) Notes

Anode

  • The anode is often a metal plate with a positive charge.
  • It attracts electrons emitted by the cathode.
  • It guides the electron beam through a small hole to form the electron beam.

Cathode

  • The cathode acts as the electron source, emitting electrons.
  • Electrons are emitted by heating a filament (e.g., tungsten) or using methods like field emission.
  • The cathode is typically a heated tungsten filament or a LaB6LaB_6 crystal.

Accelerating Voltage

  • The difference in potential (voltage) between the cathode and anode creates an electric field.
  • This electric field accelerates the electrons, giving them the energy needed for imaging.

Transmission Electron Microscope (TEM)

  • The TEM operates on the same principles as the light microscope (LM) but uses electrons instead of light.
  • Visibility with a light microscope is limited by the wavelength of light.
  • TEMs use electrons as a "light source"; their much lower wavelength makes it possible to get a resolution a thousand times better than with a LM.
  • Can see objects to the order of a few angstroms (10−10m)(10^{-10} m).
  • Can study small details in the cell or materials down to near atomic levels.
  • The possibility for high magnifications has made the TEM a valuable tool in medical, biological, and materials research.

Principle: Magnetic Lenses Guide the Electrons

  • A "light source" at the top of the microscope emits electrons that travel through a vacuum in the column of the microscope.
  • A vacuum is necessary because:
    • If the filament was surrounded by air it would quickly burn out (like a light bulb).
    • The electrons would also collide with the gas molecules and never reach the sample.
    • If gas molecules reacted with the sample, different compounds could form and condense on the sample, reducing the quality of the image.
  • The electron beam then travels through the specimen you want to study.
  • Instead of glass lenses focusing the light in the LM, TEM uses electromagnetic lenses to focus the electrons into a very thin beam.
  • Depending on the density of the material present, some of the electrons are scattered and disappear from the beam.
  • At the “bottom” of the microscope, the unscattered electrons hit a fluorescent screen, which gives rise to a "shadow image" of the specimen with its different parts displayed in varied darkness according to their density.

Preparation of Sample

  • Since TEM works in a vacuum:
    • Must fix sample (glutaraldehyde is typically used as a primary fixative; cross-links molecules with each other and trap them together to form stable structures).
    • Osmium tetroxide is used as a secondary fixative.
    • Tissue is then dehydrated in alcohol or acetone to remove all the water.
    • The specimen can be embedded in plastic that polymerizes into a solid hard plastic block.
  • Embedded tissue specimen block (embedded not in wax, but in epon resin) is cut into thin sections by a diamond knife in an instrument called ultramicrotome.
  • Each section is only 50-100 nm thick.
  • Sections are floated out on water and collected with an eyelash brush and placed on a metallic grid (copper typically used) that has been coated with a thin plastic film to lend support to sections.
  • Sections on the grid are stained with heavy metals ions, like uranium and lead, which scatter the electrons and improve the contrast in the microscope.
  • The grid is now placed in the TEM and the tissue can now be studied under the electron beam.
  • Internal structures of the cell are viewed using TEM (e.g., mitochondria, ribosomes, microtubules, membranes).
  • Examples:
    • Sperm flagellum from a caddies fly
    • Golgi apparatus from the spermatid of a dog

PRINCIPLE OF WORKING OF TEM

  • Electrons possess a wave-like character.
  • Electrons emitted into a vacuum from a heated filament with increased accelerating potential will have a small wavelength.
  • Such higher-energy electrons can penetrate distances of several microns into a solid.
  • If these transmitted electrons could be focused - images with much better resolution can be produced.
  • Focusing relies on the fact that electrons also behave as negatively charged particles and are therefore deflected by electric or magnetic fields.

ADVANTAGES & DISADVANTAGES OF TEM

Advantages

  • TEMS offer very powerful magnification and resolution.
  • 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.

Disadvantages

  • TEMS are large and very expensive.
  • Laborious sample preparation is required.
  • Operation and analysis require special training.
  • Samples are limited to those that are electron transparent.
  • TEMS require special housing and maintenance.
  • Images are black and white.

BIOLOGICAL APPLICATIONS

TEM

  • In medicine as a diagnostic tool - important in renal biopsies.
  • Cancer research - studies of tumor cell ultra structure.
  • Toxicology - to study the impacts of environmental pollution on the different levels of biological organization.