Image analysis involves processing pixel-level data from microarray images to determine the intensity of signals for each probe. After hybridization of fluorescently labeled cDNA to the microarray, a laser stimulates the array, and fluorescence intensity is measured at a dense grid of pixels, creating an image file that stores all pixel intensities. Image analysis steps include gridding, segmentation, background correction, feature intensity extraction, and flagging spots for exclusion. These steps differ between cDNA microarrays and Affymetrix GeneChip arrays. Image generation uses a scanner or CCD camera to quantify fluorescence intensity at each pixel, saving data as a 16-bit TIFF file. Fluorescent dyes (Cy3 and Cy5) emit photons at different wavelengths, which are filtered and detected by a photomultiplier tube (PMT). PMT voltage adjustment ensures balanced intensities and maximizes dynamic range. Saturated pixels, where too many photons are detected, appear as white in the image. Proper normalization minimizes signal intensity variations caused by PMT adjustments. For cDNA microarrays, raw intensities from two channels are stored as 16-bit TIFF files. These are often combined into a 24-bit RGB image for visualization, with Cy3 in green, Cy5 in red, and blue set to zero. Image analysis uses the original 16-bit TIFF files. Gridding involves overlaying a rectangular grid to isolate spots, typically automated using software that estimates array position, grid spacing, and spot spacing. GenePix, for example, automatically generates grids based on printing information and allows manual fine-tuning.Image analysis involves processing pixel-level data from microarray images to determine the intensity of signals for each probe. After hybridization of fluorescently labeled cDNA to the microarray, a laser stimulates the array, and fluorescence intensity is measured at a dense grid of pixels, creating an image file that stores all pixel intensities. Image analysis steps include gridding, segmentation, background correction, feature intensity extraction, and flagging spots for exclusion. These steps differ between cDNA microarrays and Affymetrix GeneChip arrays. Image generation uses a scanner or CCD camera to quantify fluorescence intensity at each pixel, saving data as a 16-bit TIFF file. Fluorescent dyes (Cy3 and Cy5) emit photons at different wavelengths, which are filtered and detected by a photomultiplier tube (PMT). PMT voltage adjustment ensures balanced intensities and maximizes dynamic range. Saturated pixels, where too many photons are detected, appear as white in the image. Proper normalization minimizes signal intensity variations caused by PMT adjustments. For cDNA microarrays, raw intensities from two channels are stored as 16-bit TIFF files. These are often combined into a 24-bit RGB image for visualization, with Cy3 in green, Cy5 in red, and blue set to zero. Image analysis uses the original 16-bit TIFF files. Gridding involves overlaying a rectangular grid to isolate spots, typically automated using software that estimates array position, grid spacing, and spot spacing. GenePix, for example, automatically generates grids based on printing information and allows manual fine-tuning.