Molecular Devices ImageXpress Pico, Руководство по установке

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Chapter 1: ImageXpress Pico Automated Cell Imaging System
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Dichroic Mirror
In the ImageXpress Pico System, the dichroic mirror is in a filter cube.
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 handling 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 mirror) at shorter wavelengths.
In practice, the characteristic transmission spectrum for a dichroic mirror looks similar to the
following graph.
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 also 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.