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Imaging System Hardware
Excitation
In the GenePix scanner, laser excitation is provided by individual 635 nm
and 532 nm lasers. These wavelengths correspond to the wavelengths used
to excite the fluorophores Cy3 and Cy5 (Amersham Biosciences)
respectively, which are currently the most widely used fluorescent dyes for
microarray imaging. The individual lasers have been selected because of
their superior optical performance and reliability. Since the performance of
such lasers is often sensitive to external temperature fluctuations, the
GenePix scanner uses an active temperature stabilization design to
minimize temperature-based laser fluctuations. Furthermore, each laser has
a feedback system to compensate for normal variations in laser intensity.
Laser excitation sources are often used in scanning systems instead of
conventional wide-band white light sources such as a xenon or mercury
light source. The reasons for this are quite simple: conventional wide-band
light sources do not deliver high enough photon density at the appropriate
wavelength to a sample in a scanning system. In addition, it is often
difficult to use optical filters to obtain spectrally pure, narrow-band
excitation light, which is essential for having an optimal depth of field.
Optical Path
Excitation laser light is directed onto the slide after passing through a series
of filters and mirrors. While laser light is by definition very narrow band,
the GenePix scanner employs additional optical filters to ensure that no
spurious excitation light is directed onto the slide. If the laser light
impinges on an appropriate fluorophore bound on the slide, emission light
of a longer wavelength is emitted (the difference between the emission and
excitation wavelengths is called the Stokes shift). These emission photons
are directed back through the optical system where they pass through
another set of bandpass filters (see “Emission filters”) before reaching the
photodetector.
GenePix 4100A User's Guide, Copyright 2005 Axon Instruments / Molecular Devices, Corp.