AW: AW: Leica resonant scanner-live for cell imaging

Posted by Stefan Terjung on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Leica-resonant-scanner-live-for-cell-imaging-tp590925p590932.html

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Dear Michael,

Ok, I should have installed 1.8 obviously, good to know.

If you plot fluorescence intensity against excitation intensity you get a
saturation curve. As one typically uses high power for point scanning one
easily gets into this saturation range.

This means that increasing the excitation light even more to compensate for
faster scanning you don´t gain as much fluorescence intensity (there is a
limited number of fluorophores and if (nearly) all are excited you can´t
excite more) and it´s even more likely that you bleach stronger as it´s more
likely that you excite a excited fluorophore (which significantly increases
the chance to bleach).

Regards,

Stefan

> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List
> [mailto:[hidden email]] Im Auftrag von Michael Weber
> Gesendet: Mittwoch, 28. November 2007 13:45
> An: [hidden email]
> Betreff: Re: AW: Leica resonant scanner-live for cell imaging
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Just to add that, line accumulation is available since LAS AF
> v1.8. So you can finally set it up using "Live" Scan.
>
> How does 2x Accumulation compete compared to 2x more laser
> power? The total illumination power added to one pixel is in
> the end simliar. So signal-to-noise and bleaching effects
> should be comparable as well, or not?
>
> cheers,
> Michael
>
>
> Stefan Terjung wrote:
> > - But you can get quite comparable time-lapse sequences with a bit
> >   more 'fine-tuning' on the SP5 (at least for some cases):
> > a) use accumulation (unfortunately only frame accumulation
> >          available yet) to compensate for lower brightness due to
> >   shorter pixel dwell time instead of increasing the
> laser power
> >   => this is similar to increasing the exposure time on a
> >            spinning disk without saturating the fluorophores