Fluorescence microscope is an optical microscope that uses a much higher intensity light source which excites a fluorescent species in a sample, which in turn emits a lower energy light of a longer wavelength that produces the magnified image. The property of fluorescence and phosphorescence are used for developing the image in this differentiate them from light microscopes and electron microscopy.
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (through objective). It refers to any microscope that uses fluorescence to generate an image, whether it is a simpler set-up like an epifluorescence microscope, or a complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image.
Fluorescence microscopy has become an essential tool in biology and the biomedical sciences, due to its attributes that are not readily available with traditional optical microscopy. The use of an array of fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity. In fact, this microscopy can even reveal the presence of a single molecule.
Fluorochromes are stains that attach themselves to visible or sub-visible structures, often highly specific in their attachment targeting, and have a significant quantum yield.
Various light sources are used during fluorescence microscopy. The major ones include:
- High power light-emitting diodes (LEDs)
- Xenon arc lamps or mercury-vapor lamps
- Supercontinuum sources
Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused to the detector by the objective. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light.
The fluorescence is emitted from the subject and focused through the same objective that the original light source passed. After passing objective lens, emitted fluorescence travels through an emission filter into an ocular lens and is focused onto the detector.