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Hi all,

I can't understate how helpful this discussion about training and best practices has been.  My sincere thanks go to everyone who's participated.

I’d like to put a finer point on a question about scanning speed.  Like some others I encourage users to just set the fastest speed and leave it there.  Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view.  My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power.  Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it.

As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state.  Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise.  Is that right?  Thanks!


T

Timothy Feinstein, Ph.D.
Research Scientist
Department of Developmental Biology
University of Pittsburgh