Biomedical Engineering Reference
In-Depth Information
ideal.
In the starring approach to gene expression analysis, a large swath of laser light excites many spots
in the microarray at a time (see Figure 6-9 ), producing a fluorescence pattern resembling a field of
stars—hence the name. The fluorescence pattern is captured by a photo detector, processed, and
analyzed. In starring, the major sources of variability are non-uniformity in illumination intensity and
differences in the sensitivity of the image-detection circuitry over the area of the microarray being
read. For example, because the intensity of the florescence signal is a function of the power of the
reading laser, if the power of the laser beam falls off significantly near the edges of a field, then the
level of gene expression represented by those spots will be underrepresented. Similarly, the
expression of genes represented by spots excited by the center of the beam will be overrepresented.
Even if the excitation intensity is uniform across the area of the microarray being read, the
characteristics of the image capture optics and associated circuitry can introduce artifacts in the
fluorescence signal strength because of non-uniform sensitivity to light across the area being
measured. For example, the sensitivity of the detector may vary from one edge of the detector to the
next. As a result, unless these effects are addressed in the final analysis, the gene expression figures
will be invalid.
Figure 6-9. Sources of Variability in the Starring Method of Reading a
Microarray. Not only may the laser intensity be nonlinear across the area of
the microarray that is excited by the swath of laser light, but the
photodetector may exhibit variations in sensitivity across the detector
aperture as well.
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