Fluorescent Light Path
The fluorescent microscope is used to visualize specimens that fluoresce, that is to say, emit light of a different color (always a higher wavelength) than the light absorbed by the specimen. Fluorescence occurs either because of naturally occurring fluorescent materials in the cells (such as chlorophyll and related molecules) or because the cells have been stained with a fluorochrome. Fluorochromes are stains similar to cell and tissues stains used in light microscopy and have been chosen or designed to be highly specific in their attachment to molecules in cells. The use of fluochromes has made it possible to view cells and subcellular components of cells with a high degree of specificity amid non-fluorescing material (see the DAPI stained nucleus below).
A cell nucleus stained with DAPI (courtesy Olympus Microscope Co.)
Each fluorochrome
has a specific excitation wavelength and emission wavelength and there
are many fluorochromes in use today. Thus, fluorescent microscopes are
generally equipped with several types of fluorescent filter cubes. The
most common cubes are labeled as B, G and U filter emitters. Link
to Applications of Fluorescent Microscopy for more detail on filter cubes.
FLUORESCENCE LIGHT PATH
The animation at the left shows the optical system for an epi-fluorescence upright microscope. The mercury lamp emits ultraviolet (UV) light that is directed to a specific fluorescence filter cube located in the mirror turret unit. The exciter filter in the cube is designed to allow specific wavelengths of UV light to pass though (those that are absorbed by the dye in use) which are reflected by the dichroic mirror and pass through the objective lens to illuminates the specimen (violet rays).
The light emitted from the specimen (multiple color rays) is directed upwards through the objective and pass through the dichroic mirror (which removes the incident UV light and allows the longer wavelength visible light to pass through). The light emitted is viewed after passing through the emission filter which selects which wavelength of light passes through to the eyepieces (only the green ray). In this case, one sees a bright green glowing dye-stained cell or organelle against a dark background.