Fluorescence microscopy

In microscopy, fluorescence can be used as a label or tag when preparing specific biological probes. Some biological substances, such as chlorophyll and some oils and waxes, have primary fluorescence (auto-fluorescence). But most biological molecules or structures do not fluorescence on their own, so they must be linked with fluorescent molecules fluorochromes) in order to create specific fluorescent probes.

The fluorochromes emit light of a given wavelength when excited by incident light of a different (shorter) wavelength. To view this fluorescence in the microscope, several light filtering components are needed. Specific filters are used to isolate the excitation and emission wavelengths of a fluorochrome. A dichroic beam splitter (partial mirror) reflects shorter wavelengths of light and allows longer wavelengths to pass. A dichoric beam splitter is required because the objective acts as both a condenser lens (excitation light) and objective lens (emission light); therefore the beam splitter isolates the emitted light from the excitation wavelength. This epi-illumination type of light path is required to create a dark background so that the fluorescence can be easily seen. The wavelength at which a beam splitter allows the longer wavelengths to pass must be set between the excitation and emission wavelengths of any given fluorochrome so that excitation light is reflected and emission light is allowed to pass through it.

A bright light source producing the correct wavelengths for excitation is also required for fluorescence microscopy, normally a mercury arc lamp. When using confocal microscopy to view fluorescence, where up to 95% of the emission light is filtered out, specific wavelength lasers are used, as these are extremely bright and monochromatic. Fluorescent labelling has a further advantage over other histochemical stains, in that two or more fluorochromes can be detected separately using optical filters, therefore components can be labelled specifically and identified separately in the same sample (double labelling).