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The papers are very interesting, but seem to be saying different things.
Donnert's paper basically says that giving the flurophore time to recover
helps increase signal and reduce bleaching.  Ji's paper, on the other hand
(note it is for 2p rather than confocal) states that increasing the number
of pulses per unit time achieves the same effect.  The papers in a sense
seem to contradict each other.  Any thoughts or comments, anyone?  Have
other groups verified these results?

Craig


On Sun, Oct 31, 2010 at 9:25 PM, Peng Xi <[log in to unmask]> wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Craig,
>      These two articles are very useful:
>
> Gerald Donnert, Christian Eggeling & Stefan W Hell, "Major signal
> increase in fluorescence microscopy through dark-state relaxation",
> Nature Methods - 4, 81 - 86 (2007)  doi:10.1038/nmeth986
> In this paper, the photobleaching is minimized with dark state
> relaxation (single photon excitation), which needs a low repetition
> rate (<=1MHz).
>
>
> Na Ji, Jeffrey C Magee & Eric Betzig, "High-speed, low-photodamage
> nonlinear imaging using passive pulse splitters",
> Nature Methods 5, 197 - 202 (2008)
> In this paper, by increasing the repetition rate in two-photon
> excitation, the effective excitation power is decreased, therefore a
> lower photobleaching is obtained.
>     Thank you.
>
>
> Sincerely,
> Peng Xi
> Ph. D.    Associate Professor
> Dept. of Biomedical Engineering, College of Engineering
> Peking University, Beijing, China
> Tel: +86 10-6276 7155
> Email: [log in to unmask]
> http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng>
>
> On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <[log in to unmask]>
> wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi folks.  I've been noticing a number of microscope companies have been
> > offering 'white' tunable lasers with their confocals.  Most of these
> systems
> > appear to be pulse-laser driven supercontiuum-based light sources.  My
> > question for the list is will the pulsed excitation be more effective for
> > even single photon fluorescence compared to conventional CW excitation?
>  I'm
> > thinking the relaxation time between pulses may help with photobleaching.
> > Does anyone have any thoughts or experiences to share on the matter?
> >
> > Thanks,
> >
> > Craig
> >
>