Imaging Methods

By Levi Clancy for Student Reader on
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Numerical aperture is a measure of light-gathering ability.

MethodOverview

(Compound) Light Microscope

These use visible light. Bright-field, phase-contrast, dark-field and fluorescence are the most common light microscopes. These are 2-dimensional.

Bright-Field Microscopy

Visualizes via differences in density, and lacks high contrast. Staining is usually required to visual cells using bright-field microscopy, making it worthless for most applications.

Phase-Contrast Microscopy

Used to view wet-mount preparations. Cells slow incident light and thus differ in refractive index from surroundings.

Dark-Field Microscopy

Lighting hits specimen's sides only. Light reaching lens is scattered by specimen. Specimen appears light on dark background. Best resolution of all light microscopes.

Fluorescence

Visualize fluorescing specimens.

3-Dimensional

The most common instruments for 3-dimensional imaging are differential interference contract (DIC), atomic force and confocal scanning laser.

DIC

Polarized light is split by a prism. These beams traverse the specimen and enter the objective lens where they are combined into one. Since the refractive indices are different, the image looks 3-dimensional.

Atomic Force

A stylus is positioned extremely close to the specimen, repulsive atomic forces are established and these forced are used to generate an image.

Confocal Scan

Confocal Scanning Laser Microscopy (CSLM) couples laser source to light microscope. Laser is adjusted to a vertical layer and then directed at specimen. Illumination intensity is highest at plane of focus. You can see various layers by adjusting plane of focus.

Electron Microscopy

Electron microscopes use electrons, not photons, for visualization. Instead of photos, electron micrographs are produced. Cells need to be sliced into 20-60 nm slices for visualization of internal structures.

Transmission

In a vacuum, electromagnets are used as lenses. This is used to view internal cell structure.

Scanning Microscope

Specimen is coated with heavy metal, such as gold. An e- beam goes across the specimen, and e-s scattered by the metal are collected.