Does it actually make sense to normalize fluorescence intensities to fluorescent beads (e.g. TetraSpeck)?
I have heard that some people normalize the fluorescent signal of their samples to the signal of fluorescent beads (eg. TetraSpeck) imaged either on the same slide or on a separate slide, and I'm struggling to understand the logic. First, I;ve heard of it but never saw anyone actually doing it.
The beads were never treated the same way as the sample. They weren't fixed, permeabilized, blocked, stained with antibodies, or subjected to the same mounting/clearing conditions. So how can their signal say anything meaningful about variability in my sample? Considering I'm always using the same settings in the microscope and the microscope is well calibrated.
Also that's another point - in the beads manual it says that these beads are used for calibrating the microscope, not samples. Therefore, my current understanding is that bead normalization is only valid for instrument-side variability — not for sample-side variability (staining efficiency, fixation, antibody batch, autofluorescence, RI mismatch in the sample). Is that right? Are there cases where it's reasonable to use beads beyond that, or am I missing something?
Would appreciate input from anyone who routinely does quantitative fluorescence imaging. Thanks!