Transmission Electron Microscopy (TEM) - Principle and procedure

Principle of Transmission Electron Microscopy

Transmission Electron Microscopy works by transmitting a beam of electrons through an ultra-thin specimen. As electrons interact with the specimen, the transmitted electrons form an image.
Due to the short wavelength of electrons, TEM can resolve details as small as 0.1 nm, making it suitable for studying cellular ultrastructure and material properties.

Fixation: Chemical fixatives (e.g., glutaraldehyde) are used to preserve the specimen’s structure. Osmium tetroxide is applied post-fixation to stabilize lipids and enhance contrast.
Dehydration: The specimen is passed through a graded series of ethanol or acetone to remove water.
Embedding: The specimen is infiltrated with a resin (e.g., epoxy) and polymerized to form a solid block.
Sectioning: Ultra-thin sections (50–100 nm) are cut using an ultramicrotome equipped with a diamond or glass knife.
Mounting: The sections are collected on copper grids coated with a support film.
Staining: Heavy metal stains (e.g., uranyl acetate, lead citrate) are applied to enhance electron contrast.

Vacuum System: The microscope’s column is kept under a high vacuum to prevent electron scattering by air molecules.
Electron Source: The electron gun (either thermionic or field emission) is activated to generate the electron beam.
Alignment: Electromagnetic lenses are used to align the electron beam.
Imaging: The specimen grid is inserted, and the focus and astigmatism are adjusted for a sharp image.

Detection: The image is viewed on a fluorescent screen.
Image Capture: A digital camera or photographic film is used to capture the image.

Cellular Ultrastructure: Detailed study of organelles like mitochondria and the endoplasmic reticulum.
Virology: Visualization of virus particles.
Materials Science: Analysis of crystal structures and defects.