Confocal Scanning and Artifact Rejection and Artifact Rejection
Microarray spots are very thin, usually less than 10 µm. It is the thinness of microarray spots that allows the use of confocal scanning to limit the depth of focus which prevents the imaging of many undesired background artifacts. Some imaging artifacts that are eliminated by confocal scanning include fluores-cent dust, contamination on the back surface of the sample, substrate or glass fluorescence, common air-borne dust particles (clothing fibers, skin, hand oil, etc.) and fluorescence contamination from any of the scanner's optical components.
The term "confocal" means that there are two places where the light (excitation and emission) is in focus. In a confocal scanner, excitation light is focused at the substrate and emission light focused at the pin-hole. This pinhole restricts the depth of focus of the instrument and reduces the imaging of artifacts. The laser beam enters the system and is reflected from a beamsplitter through the objective lens. The focused laser spot induces fluo-rescence, which is collected by the objective lens and converted into a parallel or "collimated" beam. That fluorescence beam travels back through the b e a m s p l i t t e r, through an emission filter to a second lens called the "detector lens". The detector lens focuses the collimated fluorescence beam, and a pin-hole aperture is placed at the focus. The detector, a P M T in the S c a n A r r a y ® micoarray analysis s y s t e m s , intercepts the light that passes through the pinhole. The beamsplitter needs to reflect laser light and transmit fluorescence light, both with high ef f i c i e n-c y. Laser light reflects from the microarray substrate and needs to be removed from the fluorescence beam, and the beamsplitter performs the primary filt e r i n g .
A point beneath the microarray substrate that is emitting fluorescence: a piece of dust, for example. The emission is collect-ed by the objective lens, but because it is not at the objective's focal point, the fluorescence beam is con-v e rging rather than collimated. The converg i n g beam then goes through the emission filter and the detector lens. Because the beam is not collimated, the detector lens focuses it in front of the pinhole. By the time the beam hits the pinhole, it has expand-ed to a much larger diameter. Most of the light from this out-of-focus point is thus blocked by the pin-hole. The same phenomenon occurs with fluorescent sources above the substrate: they create a diver g i n g fluorescence beam and the detector lens focuses behind the pinhole. Again, most of the out-of-focus light is blocked before hitting the detector.
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