Superresolution fluorescence microscopy is very sensitive to chromatic aberration. Dichroic mirrors, while being biplanar, are not normal to the optical axis and so will cause some dispersion in one lateral direction for the transmitted light.

Why are dichroics in microscopes always longpass? If we used a shortpass instead the emission light would be reflected on the first surface and will have no dispersion. I don’t believe they can suffer from ghosting like NPBS plates due to the very high reflecting efficiency for the reflected wavelengths.


1 Answer 1


Using a short-pass filter arrangement might reduce aberration but increase background and require more care not to damage the mirror.

As Thorlabs and Leica tutorials explain, long-pass dichroic mirrors in typical fluorescence microscopes are used to reflect the short wavelength excitation radiation onto the sample while allowing the returning longer wavelength fluorescence radiation to pass through the mirror to the imaging detector. A general rule for dichroic mirrors is that the coated reflective surface has to be the first surface that the light hits before being reflected or transmitted. This is true for both the excitation and fluorescent radiation in this case, as shown in the left sketch below.

Light paths for long-pass and short-pass arrangements

The simplest design using a short-pass dichoric mirror requires swapping the light source and detector as shown on the right above, with the short-pass dichroic mirror passing the short wavelength excitation radiation through to the sample and reflecting the long wavelength fluorescence onto the detector.

A problem with this arrangement is that the excitation light enters the dichroic mirror backwards and passes through the mirror substrate. The excitation illumination is much more intense than the fluorescence, so the small fraction of the illumination that scatters while passing through the substrate may produce a significant background observable in the detector and (depending on the illumination intensity) may heat up and possibly distort or damage the mirror.

If you really want to use a short-pass mirror, instead of using these confocal designs, you could perhaps excite the sample with a laser whose light does not pass through the mirror, as happens with total internal reflection fluorescence microscopy.

For why a mirror is used at all, see "Why do fluorescent microscopes employ a dichromatic mirror?"


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