Widely used in the past, the technique of exciting fluorescence with transmitted light has now been replaced by incident light excitation.
Leica Incident Light Fluorescence Illuminators accommodate up to eight filter cubes containing the excitation filters, dichromatic mirrors and suppression filters.
The excitation filter is designed to have the highest possible transmission at those wavelengths which cause fluorescence in a particular specimen, while suppressing all remaining irradiation.
The dichromatic mirrorreflects the short wavelength excitation light onto the specimen, but is transparent to the longer wavelength fluorescence.
The suppression filter absorbs the excitation light reflected from the specimen which re-enters the objective, but is highly transparent to the fluorescence wavelengths specific to the specimen.
The interaction of the three filter components results in a bright, contrasty fluorescence image against a dark specimen background.
This combination of perfectly matched filters in a cube, which can easily be exchanged in a matter of seconds, explains the great ease of use of Leica incident light fluorescence systems, and is an essential criterion for the standardization of immunofluorescence tests.
Incident light fluorescence microscopy has now become indispensable in clinical and biological laboratories.