Methods used in study of cells and tissues

Microscopes use lenses to magnify images created by photons or electrons.

Bright-field

 * Visible light is directed at a sample from below - condenser focuses light onto sample
 * Light is transmitted through the tissue, enlarged and projected by the objective lens
 * Eyepiece further magnifies image and directs toward viewer
 * Immersion oil is used at higher magnifications (1000X) to increase the resolving power; it keeps excess light waves from reflecting
 * Most simple and common method

Fluorescence Microscopy

 * UV light is directed at sample
 * Light emitted from tissue is within visible spectrum
 * Used to localize particular macromolecules within cell (ex: DNA, antibodies)

Phase contrast

 * Light passing through different structures in the sample changes speed accordingly
 * Lens system visualizes these changes as lighter/darker areas
 * Advantage: does not require fixation/staining, so it can be used to view living cells

Confocal microscopy

 * Aligns the point light source, focal point of lens, and pinpoint aperture of detector in one focal plane
 * Reduces stray light beams normally present in bright-field microscopy
 * Image is sharper and of higher resolution

Polarizing Microscopy

 * Recall that visible light has electric and magnetic components that are perpendicular, but can be polarized by crystalline substances
 * The sample is placed between two perpendicular filters; this normally cancels out all transmitted light
 * However, if the sample also contains substances of a periodic structure (ex: collagen), they will rotate the axis of light after it passes through the first filter, and it will no longer be perpendicular to the second filter
 * The image formed shows the substance on a black background

Transmission Electron Microscopy (TEM)

 * Electrons pass through sample and are focused via electromagnetic "lenses"
 * Denser substances absorb/deflect electrons better and appear darker
 * Image contrast improves with the use of heavy metal ions
 * Microstructural details are better observed with cryofracture/freeze etching
 * Typical magnification is up to 120,000 X

Scanning Electron Microscopy (SEM)

 * Sample is coated with a heavy metal
 * Electrons reflect off of sample at various angles
 * Image appears 3D with shadows and highlights

Autoradiography

 * Radioactive monomers are offered to a culture
 * As cells synthesize polymers, they aggregate and become visible
 * Allows one to localize synthesis of biopolymers (often DNA or proteins)

Enzyme histochemistry

 * Enzyme substrate is offered to a tissue section
 * Enzymatic reaction produces product, which in turn reacts with marker compound
 * Marker compound precipitates in sites of high activity
 * Ex: used to study phosphatases, dehydrogenases, peroxidases

Immunohistochemistry

 * Antibodies (tagged for visibility) are added to tissue
 * Antibody binds only to specific protein; shows location after rinsing
 * Can be used to diagnose specific tumors or viral infections