Scanning:
Producing the microarray images
Scanning is performed using the Genepix 4000B dual laser scanner (Axon). One 16-bit TIF-format greyscale image is generated per channel for each hybridised microarray. The fluorescent spot signal is then quantified with publicly available software, as described below. False colour images are only to be used for presentations or for simple qualitative quality control checks, e.g. sub-grid alignment, spot structure, background signal, etc.
Grey scale and colour images
Spot signal saturation
Microarray scanners have a pixel intensity range of between 1 (20) to 65535 (216 - 1), inclusive. Image brightness can be changed by choosing a different PMT gain. We try to reduce the amount of saturated spots, however, there is a trade-off between having few saturated spots and losing low intensity spots, when scanning at lower PMT gains. Within some of your analysis files, spots with raw intensities greater than a fixed threshold, e.g. >65000, will be flagged as saturated.
