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Regarding the discussion on how big the spot of laser illumination is at the
sample:
I like theoretical talk too, but an empirical answer can be had by
overexposing(photobleaching) a solid mount media with a stationary,
single-point scan, and then do  a z-series to see and measure the profile of
the photobleached region. This is on my list of things to do.
As an aside, being off the optical axis can alter the results significantly.
 
 --Jeff Reece,  [log in to unmask]
 
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