Following hybridization, the levels of fluorescence from each spot for both Cy3 and Cy5 are determined using a confocal scanner. A false color image is produced from a prescan of the array; the image represents the level of fluorescent intensity using a color gradient and is solely for easy observational analysis, allowing researchers to check on the internal controls. If the negative controls are negative, the positive controls are positive, and the background is not too imposing, a gain at which the array will be scanned can be set. The gain refers to the laser power used by the confocal microscope to excite the fluorophores without inducing saturation, and is expressed as the ratio of output (intensity) to input (laser power). It is important that the slide be scanned at a gain just below the threshold where any of the spots become saturated, otherwise these will be of no use. Conversely, if the signal intensity for any probe of the spots is too low, they may be confused with the background and be unreliable. A crude observational analysis of the false color image is enough to ascertain what gain to use. Often, the array is scanned a second time at the maximum gain allowed to clarify the reading from spots with very low intensities.

The array is scanned under two wavelengths (or channels), 635 nm for Cy5 and 532 nm for Cy3, and for each dye the computer produces a separate monochromatic image (as a TIFF - tagged information file format - file) that reflects the fluorescent intensities on a black and white gradient. Once the slide has been scanned, the monochromatic TIFF images for each dye can be exported into a piece of image analysis software that performs the quantification of the fluorescence intensity.

0 0

Post a comment