Electron Microscopy Overview

Electron Microscopy

• There are two main types of electron microscopes: Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)
• TEM has a resolution power of around 0.2nm and a magnification power of up to 400,000 times.
• TEM uses a beam of electrons focused by electromagnetic lenses to create an image.
• TEM images appear as shades of black and white, with electron-dense areas appearing black and electron-lucent areas appearing white.
• Electron-lucent areas allow electrons to pass through easily, while electron-dense areas absorb or deflect electrons
• TEM uses ultrathin tissue sections (40-90nm)
• To view a TEM image, a fluorescent screen is needed to convert the energy of electrons into light
• SEM uses a beam of electrons to scan the surface of a specimen coated with a heavy metal (gold)
• The reflected electrons are collected by a detector to create a 3D black and white image on a TV screen.
• SEM only shows the surface of a specimen because the electron beam does not pass through it.

Light Microscopy

• Microscopes are used to view specimens too small to be seen with the naked eye.
• Magnification refers to the degree of enlargement, and resolution refers to the ability to show details clearly.
• The most important units of measurement in histology are:
  • centimeter (cm): 10 millimeters (mm)
  • millimeter (mm): 1000 micrometers (µm)
  • micrometer (μm): 1000 nanometers (nm)
  • nanometer (nm): 10 Angstroms (Ao)
• Light microscopes use light to illuminate the specimen.
• Electron microscopes use a beam of electrons to illuminate the specimen.
• Brightfield microscopy, phase contrast microscopy, fluorescence microscopy, polarizing microscopy, and confocal microscopy are all types of light microscopy
• TEM and SEM are types of electron microscopy.

Components of a Light Microscope

• The frame consists of the base, arm, and stage.
• The magnifying system consists of the lenses.
• The illumination system provides the light source, which can be a mirror to reflect daylight or an electric lamp.
• The condenser is located under the stage and collects and focuses a cone of light to illuminate the tissue slide.
• The objective lens enlarges and projects the illuminated image of the object in the direction of the ocular lens.
• The ocular lens (eyepiece) further magnifies the image projected by the objective lens and projects it onto the viewer's eye.

Microscope Magnification

• To calculate the total magnification of a microscope: multiply the magnification of the ocular lens by the magnification of the objective lens.
• The objective lenses provide higher magnification and resolving power than the ocular lens.
• The ocular lens only enlarges the image already obtained by the objective lens, it does not improve resolution.
• The total magnification of a light microscope depends on the objective lens used.

Special Types of Light Microscopes

• Phase contrast and interference microscopes allow the study of unstained cells, which are transparent and colorless.
• Fluorescence microscopes use fluorescent dyes to visualize specific structures or molecules.
• Polarizing microscopes are used to study crystals and substances with repeating molecular arrangements, such as collagen, microtubules, and microfilaments.
• Confocal microscopes use a laser to create high-resolution, sharp, 3D images.