Fluorescence microscopy

Fluorescent compounds (which at the exciting wavelength absorb light and then emit it at the emission wavelength) are associated to a secondary antibody which binds to the primary antibody that is itself particular for the subcellular structure under investigation in fluorescence microscopy. At the exciting wavelength upon illumination, the fluorescent compound emits light, revealing where the primary antibody has bound.

The light microscope is adapted to detect the light emitted In fluorescence microscopy, by a fluorescent compound that is utilized to stain selectively components within the cell.  if a chemical absorbs  light  at one wavelength  (the excitation  wavelength)  and then emits light at a longer wavelength (the emission wavelength) it is called fluorescent.  Two compounds which commonly used in fluorescent microscopy are and Texas red and rhodamine which emit red light, and fluorescein,  which  emits  green  light.  First, an antibody against the antigen of interest (so-called primary antibody;) is added to the specimen. A fluorescent compound is chemically coupled to a secondary antibody that identifies the primary antobody. Then the fluorescently-tagged secondary antibody is added to the tissue section or permeabilized cell, and with light at the exciting wavelength the specimen is illuminated. In the specimen the structures to which the antibody has bound may then be visualized. Fluorescence microscopy may also be applied to living cells, which permit the movement of the cells and structures within them to be followed with time.

Labeling of protein for fluorescent microscopy with a fluorescently-tagged antibody. The primary antibody recognizes the antigen of interest and binds to it in the specimen. Numerous molecules of the secondary antibody bind to the primary antibody providing amplification of the signal. Secondary antibody is covalently coupled to a fluorescent dye that emits light when illuminated at its excitation wavelength.