Imaging System Hardware
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Emission Filters
Light from the slide is filtered before reaching the photodetectors, to ensure
that as far as possible, the detectors measure only emission photons from
the appropriate fluorescent dye. The filter’s ‘window’ is matched as
closely as possible to the peak emission spectrum of the selected dye to
maximize acceptance of the desired emission light and at the same time
exclude stray laser excitation light, and light emitted by non-target
fluorescence (autofluorescence, reagent background etc.).
The GenePix scanner comes with two emission filters installed; one
optimized for the emission spectrum of the dye Cy5, and the other
optimized for Cy3. The factory-installed Cy5 (‘red’) filter is classified as
670DF40—meaning a Discriminating Filter, with pass-band centered on a
wavelength of 670 nm, and full bandwidth of 40 nm. This is ideal for
selecting the emission photons of the red dye Cy5, which has an emission
spectrum centered on 667 nm, and excluding light from the 635 nm laser
and the Cy3 dye. Likewise, the Cy3 (‘green’) filter is classified as
575DF35. The spectra for the emission filters in the GenePix scanner have
very sharp cut-on and cut-off wavelengths, so the transmission coefficient
is close to 1 within the entire specified band, and close to zero outside it.
You might have purchased additional filters for your scanner—particularly if
you use dyes with emission spectra different from Cy3 and Cy5. The
GenePix Personal 4100A scanner has capacity for six custom filters in
addition to the standard factory filters. If you decide to use different dye
and/or filter combinations, it is important to match the optical characteristics
of filter, dye, and laser. The pass-band of the filter should be centered as
close as possible to the emission peak of the chosen dye, and must be narrow
enough to exclude light from the laser, other dyes, etc., but not so narrow that
too many emission photons are excluded, otherwise sensitivity will be lost.
Photodetection
The GenePix 4100A uses a high-sensitivity, low-noise photo multiplier
tube (PMT) to detect the emitted fluorescent light. PMTs are optical
Chapter 4
