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ImageXpress Confocal HT.ai High-Content Imaging System User Guide
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Dichroic Mirror
In the ImageXpress Confocal HT.ai system, framed dichroic mirrors are in a five-position filter
wheel. See
Figure 1-2 .
A dichroic mirror is a specially designed beam splitter that transmits light above a certain cutoff
wavelength and reflects light at shorter wavelengths. This is the essential component that
allows the construction of an epi-illumination fluorescence imaging system in which the
illumination and imaging optical paths overlap at the objective lens. The same objective lens is
used to focus the illumination light on the sample as well as collect the emitted fluorescent
light to form the image.
In the illumination path, the dichroic mirror reflects shorter wavelengths from the light source up
through the objective onto the specimen.
In the imaging optical path, longer wavelength fluorescence light emitted by the excited
fluorophores in the specimen is collected by the objective lens and transmitted through the
dichroic mirror to the camera. Incident light from the sample that is shorter wavelength than the
cutoff (mostly reflected illumination light from the sample) is reflected by the dichroic mirror
(and further blocked by the emission filter), preventing it from entering the imaging system of
tube lens and camera.
The optics in a filter cube are interference filters made by depositing thin film coatings on a
glass support. These components are delicate and can be easily damaged. Always use care
when you handle a filter cube.
Dichroic Transmission Spectrum
An ideal dichroic mirror would have an infinitely sharp cut-off. That is, it would have unity
transmittance coefficient at wavelengths longer than the cut-off, and zero transmittance (and
therefore unity reflectance in a non-absorbing dichroic single-pass mirror) at shorter
wavelengths. In practice, the characteristic transmission spectrum for a dichroic mirror looks
like the following graph.
Figure 1-3: Transmission Spectrum for a Dichroic Mirror
In principle, the cutoff wavelength (or midpoint of the cutoff region) of the dichroic mirror
should be chosen to lie halfway between the absorption and emission peaks of the chosen
fluorochrome, as this simultaneously maximizes the amount of excitation light available at the
sample and the amount of collected fluorescence emission that is transmitted to the camera. In
practice, however, additional considerations such as fluorochrome efficiency can dictate that
the cutoff region is biased toward one peak or the other. This allows, for example, greater
transmission of longer wavelength image photons at the expense of less reflection of shorter
wavelength excitation light.