Instrument Components
26
5000450 D
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 4000B Microarray Scanner employs
additional optical filters to make sure 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. These emission photons are directed back through the optical
system where they pass through another bandpass filter before
reaching the photodetector.
One problem faced by dual scanning systems is crosstalk, where the
two different wavelengths of light are not sufficiently separated in the
scanning system. For example, emission light from one fluorophore can
be recorded by the detector for the second fluorophore, or spurious
excitation light for the first fluorophore can bleed back in into the
detector for the second fluorophore. Crosstalk is often a concern when
using a pair of fluorophores that have similar spectral properties. The
result can be an erroneous contribution of fluorescence from one optical
channel to the other.
The GenePix 4000B Microarray Scanner employs two approaches to
reduce crosstalk to negligible levels. First, the filter sets and excitation
lasers have been carefully chosen to ensure that crosstalk between the
two fluorophores is minimized. Second, and more importantly, the
patented optical design guarantees that the light paths of the two
channels are spatially separated.
The GenePix 4000B Microarray Scanner includes a user-controlled
focusing feature. The focal plane at the zero position is at the surface of
the glass slide, with a total depth of focus of 64
μ
m, equally distributed
on both sides of the focal plane (depth of focus is defined as the range
over which the signal is within 50% of the maximum signal). The
GenePix Pro Software allows you to set the focal plane from –50
μ
m to
+200
μ
m relative to the zero position. Positive positions refer to focal
planes above the microarray surface while negative positions are within
the slide.
Emission Filters
The GenePix 4000B Microarray Scanner is equipped with two lasers.
Each laser (red and green) includes a matching pre-installed emission
filter.
•
Standard red (660 nm to 690 nm; 670DF40)
•
Standard green (558 nm to 593 nm; 575DF35575)
Each emission filter is optimized for the emission spectrum of the
respective dye, designed to reject laser light at OD 8 (optical density)
for each respective laser excitation, and reject broad-spectrum light at
OD 5.
GenePix_4000B.book Page 26 Friday, October 22, 2010 3:07 PM
