The chapter discusses the process of image analysis in the context of gene expression microarrays. After hybridization, the array is stimulated with a laser to measure fluorescence intensity at various pixel locations, creating an image file. Image analysis involves processing pixel-level data to convert it into intensity measures for probes. The main steps include gridding, segmentation, background correction, feature intensity extraction, and flagging spots for exclusion. The methods for cDNA microarrays and Affymetrix GeneChip™ arrays differ, so these steps are discussed separately. **4.1 Image Generation** A scanner or CCD camera quantifies fluorescence intensity at each pixel, saving the data in a 16-bit TIFF file. The emitted photons from fluorescent dyes (Cy3 and Cy5) are filtered and converted into electrical signals by a PMT detector. Saturated pixels appear as white and can affect results, but proper normalization can mitigate this. **4.2 Image Analysis for cDNA Microarrays** - **4.2.1 Image Display**: The raw intensities are saved as 16-bit integers and can be displayed as 24-bit RGB images. For analysis, the original 16-bit TIFF files are used. - **4.2.2 Gridding**: The first step in analysis involves overlaying a grid to isolate each spot within a cell. This is often semi- or fully automated, using robotic spotting information to estimate grid positions and spot spacing. Software like GenePix automatically generates grids and allows manual fine-tuning.The chapter discusses the process of image analysis in the context of gene expression microarrays. After hybridization, the array is stimulated with a laser to measure fluorescence intensity at various pixel locations, creating an image file. Image analysis involves processing pixel-level data to convert it into intensity measures for probes. The main steps include gridding, segmentation, background correction, feature intensity extraction, and flagging spots for exclusion. The methods for cDNA microarrays and Affymetrix GeneChip™ arrays differ, so these steps are discussed separately. **4.1 Image Generation** A scanner or CCD camera quantifies fluorescence intensity at each pixel, saving the data in a 16-bit TIFF file. The emitted photons from fluorescent dyes (Cy3 and Cy5) are filtered and converted into electrical signals by a PMT detector. Saturated pixels appear as white and can affect results, but proper normalization can mitigate this. **4.2 Image Analysis for cDNA Microarrays** - **4.2.1 Image Display**: The raw intensities are saved as 16-bit integers and can be displayed as 24-bit RGB images. For analysis, the original 16-bit TIFF files are used. - **4.2.2 Gridding**: The first step in analysis involves overlaying a grid to isolate each spot within a cell. This is often semi- or fully automated, using robotic spotting information to estimate grid positions and spot spacing. Software like GenePix automatically generates grids and allows manual fine-tuning.