Most cellular components are colorless and cannot be clearly distinguished under a microscope. The basic premise of fluorescence microscopy is the staining of components with dyes.
Fluorescent dyes, also known as fluorophores or fluorescent dyes, are molecules that absorb excitation light of a given wavelength (usually UV) and emit light of a longer wavelength after a brief delay. The delay between absorption and emission is negligible, typically on the order of nanoseconds.
The emitted light can then be filtered from the excitation light to reveal the location of the fluorophore.
Fluorescence microscopy uses more intense light to illuminate the sample. This light excites fluorescent material in the sample, which then emits light at a longer wavelength.
The resulting image is based on the emission wavelength of a second light source or fluorescent substance other than the light originally used to illuminate and excite the sample.
processing
Light at the excitation wavelength is focused on the sample through the objective lens. The fluorescence emitted by the sample is focused on the detector through the objective lens. Since most of the excitation light is transmitted through the sample, only the reflected excitation light reaches the objective along with the emitted light.
form
"Fluorescence microscopy" refers to any microscope that uses fluorescence to generate images, whether it is a simpler device such as an epi-fluorescence microscope, or a more complex design such as a confocal microscope, which uses optical sectioning to obtain better resolution. Fluorescence image.
Most fluorescence microscopes used are epifluorescence microscopes, where excitation of fluorophores and detection of fluorescence are accomplished through the same optical path (i.e., through the objective lens).
