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| Light is assorted: filters and filter sets |
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The graph on the left shows the spectral properties in (single) fluorescence. The fluorochromes absorb light ( 1) in a narrow spectral range and emit it into a range of a longer wavelength ( 2). The Stokes shift lies between the two.
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| Spectral position of excitation (left) and emission (right) in fluorescence. |
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In fluorescence microscopy, filter combinations are arranged in the reflector area between the objective and the tube lens. These combinations determine the beam path of the excitation and emission light.
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| Function of the filter combinations |
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The exciter filter (A) filters almost monochromatic light
(2) out of the light source radiation (1). The properties of the dichroic beam splitter (B) are fascinating: it reflects the short-wave excitation light to the objective almost without any loss, but allows the fluorescence light (3) returning from the specimen via the objective to pass through almost completely.
At the same time, most of the excitation light is reflected again and can therefore no longer affect the formation of the intermediate image. Above the beam splitter, the emission light and the remainder of the excitation light hit the barrier filter (C). Only the fluorescence light can pass the filter almost unhindered because its wavelengths are longer than those of the excitation light.
In most cases, modern fluorescence filters are combinations of color lenses and interference filters – with the exception of some classical longpass filters. The interference filters have been given coatings precisely adapted to the wavelengths of the light. Such filters can be “tailor-made” and exactly meet the spectral requirements made on a fluorescence dye.
Typical spectral curves of the transmission T of filters for fluorescence microscopy:
1 Shortpass filter
2 Longpass filter
3 Bandpass filter
4 Narrowband filter
Typical, individual spectral curves for the components of the filter combination: A: Exciter filter B: Beam splitter C: Emission filter.
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