*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****
Hi,
as I understand it, in Donnert's paper the assumption is that bleaching
(especially due to action of the STED laser) is due to excitation of
triplet states of the fluorophores, which have a lifetime of several µs
and are populated by intersystem crossing from the fluorescent S1 state.
So the idea is to let the triplet states relax back to the S0 state,
either by reducing laser rep rate or faster scanning.
I haven't read Ji's paper, but in 2P work you should have photon
energies low enough that excitation from the S1 or T1 states is not of
major concern. On the other hand side, the pulse lengths in 2P are much
shorter (~200 fs) than in pulsed STED (~200-300 ps), so you could direct
excitation S0->Sn via 3P absorption or other higher-order effects.
Reducing exciation power and incresing pulse rate should give you the
same number of photons (ie SNR) with lower probability of higher-order
effects. Increasing pulse length on the other hand side would reduce the
excitation probability for 2P as well.
As stated, I haven't read Ji's paper, but that's what would make sense
to me.
Christian
Am Montag, den 01.11.2010, 16:20 -0600 schrieb Craig Brideau:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:
>
> > *****
> > 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
> > >
> >
--
Dr. Christian Schumann
INM
Leibniz-Institut für Neue Materialien gGmbH
Campus D2 2
66123 Saarbrücken
Telefon: +49 681 9300-327
Telefax: +49 681 9300-223
E-Mail: [log in to unmask]
Homepage: www.inm-gmbh.de
------------------------------------------------------------------------
Sitz der Gesellschaft: Saarbrücken
Rechtsform: gGmbH
Amtsgericht Saarbrücken, HRB 8525
Geschäftsführer: Prof. Dr. Eduard Arzt (Vorsitz),
Prof. Dr. Michael Veith, Dr. Roland Rolles
Kuratoriumsvorsitzender: StS Peter Hauptmann
USt.-ID: DE 138167776
------------------------------------------------------------------------
|
