I am measuring fluorescence intensity using a Tecan microplate reader from two assay plates with the same samples starting at two different time points. I want to plot both the plate readings in the same graph. So, should I consider 'OPTIMAL' gain or 'MANUAL' gain values for this?
Manual. Use the same manual gain setting for all measurements if you want to be able to compare them. If you choose Optimal, the gain may differ between measurements, making it impossible to compare them.
The Manual gain should be selected based on a sample containing what may be expected to be the highest fluorescence intensity, leaving some room for higher intensities. You don't want to get too close to saturating the detector, so a gain that gives about one-third of detector saturation with the brightest sample is a reasonable choice.
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