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The Affymetrix GeneChip Scanner 3000,,, New Advances in Scanner Design for Superior Performance, Reliability, and Dynamic Range

optical structure has three main limitations. First, imaging performance is characterized by geometric distortion and non-uniform emission collection. The visible manifestations of these effects are seen as inaccurate gridding and non-uniform imaging intensity. Non-ideal optics and open-loop, scanmotion control are the principle causes of geometric distortion. Lens aberrations and optical misalignment lead to non-uniform emission collection. Second, the low numerical aperture (NA) of the objective lens places a constraint on achievable scan speed. NA, which is related to distance from the objective to the sample (Figure 1), is a measure of the optical collection efficiency of the objective lens, i.e., the ability to collect fluorescent photons. Simply put, due to the low NA, the detector needs to dwell on the biological sample site for a relatively long time in order to acquire sufficient photons to ensure acceptable signal-to-noise performance. The previous GeneChip array scanner addressed collection efficiency by averaging two or more successive lines, or successive images, to reduce the effects of signal noise. Third, emission path alignment is difficult. Photons from the fluorescent emission are collected along a line that coincides with the excitation line-scan. The returning emission beam is collected by the objective lens, passed through an emission filter, and re-focused to a small spotactually a line since the beam is scanning. The beam then passes through a slit that serves a function analogous to the pinhole in a conventional confocal microscope, i.e., rejection of light emitted or scattered into the objective from planes other than the focus plane. Precise alignment of the emission line (a result of the scanned emission beam) with the slit over the entire width of the scan is difficult, leading to another source of non-uniform collection efficiency across the image field. It is not uncommon to have systematic intensity gradients of 15% or mor
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