A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption.
In most cases, a component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore (such as Green fluorescent protein, fluorescein or DyLight 488). The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (Xenon or Mercury arc-discharge lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter (see figure below). The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.
Most fluorescence microscopes in use are epi-fluorescence microscopes (ie : excitation and observation of the fluorescence are from above (epi) the specimen). These microscopes have become an important part in the field of biology, opening the doors for more advanced microscope designs, such as the confocal laser scanning microscope and the total internal reflection fluorescence microscope (TIRF).
Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care must be taken to prevent photobleaching through the use of more robust fluorophores or by minimizing illumination.
Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in Life Sciences. In this process, instead of transmitting the excitatory light through the specimen it is passed through the objective onto the specimen. Since only reflected excitatory light filters through, the transmitted light is filtered out, giving a much higher intensity. Fluorescent or fluorochrome stains are applied to the specimen to provide an estimated count.
It can be used to find routine direct total counts of bacteria in water samples.
- Bradbury, S. and Evennett, P., Fluorescence microscopy., Contrast Techniques in Light Microscopy., BIOS Scientific Publishers, Ltd., Oxford, United Kingdom (1996).
- Rost, F. and Oldfield, R., Fluorescence microscopy., Photography with a Microscope, Cambridge University Press, Cambridge, United Kingdom (2000).
- Principles of Fluorescence
- Nikon MicroscopyU, tutorials from Nikon
- Fluorophores.org - The database of fluorescent dyes