Scanning

As a general rule we like to have around 10% of the spots at the maximum intensity (i.e., saturated). This applies when using both organic dyes and Q-dots for visualization of bound antibody. For regular laser scanners, the laser power (LP) is usually kept constant and then we adjust the photomultiplier tube (PMT) output to achieve ~10% saturation. The LP you use depends on your scanner. For example, we use 10o% LP on our Genepix. For the ArrayCAM [finish this].

Once the settings are ideal, we usually do not like to change them. However, in some cases you may need to adjust the PMT, particularly when scanning slides probed on different days. Doing experiments on different days is not recommended since inter-experimental differences may be introduced, but unavoidable if the numbers of sera are too large to be probed all at once. Or of you need to repeat something down the road. Scan everything at the same settings first. When you do a scatter plot of the data of the reference positive control serum probed on different days, ideally the slope should = 1. Any large deviations from this may require re-scanning one set of slides at a different PMT to bring the slope closer to 1. Small  deviations may be evened out in the existing data set using the “fold-over control” (FOC) type of normalization (check the effect FOC normalization has on the slope).