Two filters are typically used in fluorescence microscopy:
- An illumination (excitation) filter – ensures the illumination is near monochromatic and at the correct wavelength.
- Second emission (detection) filter – ensures none of the excitation light source reaches the detector.
So how does a fluorescence microscope work?
The specimen is illuminated with light of a specific wavelength that is absorbed by the fluorophores, causing them to emit light of longer wavelengths (in other words a different color than the absorbed light). The illumination light is separated from the weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope include:
- Light source (xenon arc lamp or mercury-vapor lamp)
- Excitation filter
- Dichroic mirror (or dichromatic beamsplitter)
- Emission filter
Most fluorescence microscopes are “epi-fluorescence microscopes”. Epi refers to the fact that the excitation and observation of the fluorescence are from above the specimen.
Fluorescence microscope image of mouse embryo.