Fluorescence is one of the most commonly used physical phenomena in biological and analytical microscopy, mainly because of its high sensitivity and high specificity. Fluorescence is a form of luminescence. Fluorescence microscopy even allows users to determine the distribution of a single molecule species, its amount and its localization inside a cell. Colocalization and interaction studies can be performed, and ion concentrations as well as intra- and intercellular processes like endocytosis and exocytosis can be observed. With the help of super-resolution fluorescence microscopy, it is even possible to image sub-resolution structures.
Fluorescence microscopes used in research applications are based on a set of optical filters:
- an excitation filter
- a dichroic beam splitter and
- an emission filter
The filters are often plugged in together in a filter cube (compound microscopes) or in a flat holder (mainly stereo microscopes).
Whereas the excitation filter selects the wavelengths to excite a particular dye within the specimen, the emission filter serves as a kind of quality control by letting only the wavelengths of interest emitted by the fluorophore pass through. The dichroic mirror's purpose is to reflect light in the excitation band and transmit light in the emission band, enabling the classic epifluorescence incident light illumination.
Leica Microsystems’ fluorescence stereo microscopes use the TripleBeam technology, a separate (third) beam path for fluorescence illumination of the sample without a dichroic mirror.
Therefore one excitation filter for the illumination beam path and two emission filters, one for each observation beam path, are needed.