Pulsed vs CW laser sources for single-photon fluorescence

[Craig Brideau]

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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

[Peng Xi-2]

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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: [phone]
Email: [hidden email]
http://bme.pku.edu.cn/~xipeng

On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <[hidden email]> 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
>

[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 <[hidden email]> 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: [phone]
> Email: [hidden email]
> http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng>
>
> On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <[hidden email]>
> 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
> >
>

[Christian Schumann]

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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 <[hidden email]> 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: [phone]
> > Email: [hidden email]
> > http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng>
> >
> > On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <[hidden email]>
> > 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: [phone]
Telefax: [phone]
E-Mail: [hidden email]
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
------------------------------------------------------------------------

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks for your insight, Christian.  So what it boils down to is that for
single photon interactions the primary method of damage is intrasystem
crossing into triplet states.  So the solution is to allow time for these
states to relax.  Conversely, for two photon the primary damage mechanism
are unwanted 3rd order or higher effects so lowering peak energy is the way
to go.  I'm just curious how much variability there is depending on your
sample.  Would triplet state excitation also be a factor at all in 2p, or
are the photon energies really low enough?  After all, couldn't 2p
interactions excite to triplet states as well?  Or is there just not enough
energy to cause crossing?

Craig


On Tue, Nov 2, 2010 at 2:15 AM, Christian Schumann <
[hidden email]> wrote:

> *****
> 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 <[hidden email]> 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: [phone]
> > > Email: [hidden email]
> > > http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng> <
> http://bme.pku.edu.cn/%7Exipeng>
> > >
> > > On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <
> [hidden email]>
> > > 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: [phone]
> Telefax: [phone]
> E-Mail: [hidden email]
> 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
> ------------------------------------------------------------------------
>

[Christian Schumann]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Craig,

I don't know if there is much data out there on the 2P excitation cross
sections of triplet states, but I'd guess that these are probably not
really high.
Excitation directly to the triplet state is quantum mechanically not
allowed (at least for one-photon transitions) due to the unequal spin
multiplicities, so the cross section is practically non-existent. I'd
intuitively say that it's even lower for a 2P transition.
The only way to get to the triplet state is by intersystem crossing,
which is mostly based on spin-orbit coupling and depends a lot on the
specific molecule and environment. But perhaps some FCS people have
measured triplet population ratios after 2P excitation.

I might be terribly wrong, my spectroscopy days were quite a while back,
as was my QM course. Probably anyone else has some more input on this.

Christian

Am Dienstag, den 02.11.2010, 11:48 -0600 schrieb Craig Brideau:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks for your insight, Christian.  So what it boils down to is that for
> single photon interactions the primary method of damage is intrasystem
> crossing into triplet states.  So the solution is to allow time for these
> states to relax.  Conversely, for two photon the primary damage mechanism
> are unwanted 3rd order or higher effects so lowering peak energy is the way
> to go.  I'm just curious how much variability there is depending on your
> sample.  Would triplet state excitation also be a factor at all in 2p, or
> are the photon energies really low enough?  After all, couldn't 2p
> interactions excite to triplet states as well?  Or is there just not enough
> energy to cause crossing?
>
> Craig
>
>
> On Tue, Nov 2, 2010 at 2:15 AM, Christian Schumann <
> [hidden email]> wrote:
>
> > *****
> > 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 <[hidden email]> 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: [phone]
> > > > Email: [hidden email]
> > > > http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng> <
> > http://bme.pku.edu.cn/%7Exipeng>
> > > >
> > > > On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <
> > [hidden email]>
> > > > 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: [phone]
> > Telefax: [phone]
> > E-Mail: [hidden email]
> > 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
> > ------------------------------------------------------------------------
> >

--
Dr. Christian Schumann
INM
Leibniz-Institut für Neue Materialien gGmbH
Campus D2 2
66123 Saarbrücken

Telefon: [phone]
Telefax: [phone]
E-Mail: [hidden email]
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
------------------------------------------------------------------------

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Here's a question: Would the spin of the incident photons influence
intersystem crossing?  As I understand it, crossing requires angular
momentum changes.  If the incident photon possesses + or - spin would it
make a difference?

Craig


On Tue, Nov 2, 2010 at 12:06 PM, Christian Schumann <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Craig,
>
> I don't know if there is much data out there on the 2P excitation cross
> sections of triplet states, but I'd guess that these are probably not
> really high.
> Excitation directly to the triplet state is quantum mechanically not
> allowed (at least for one-photon transitions) due to the unequal spin
> multiplicities, so the cross section is practically non-existent. I'd
> intuitively say that it's even lower for a 2P transition.
> The only way to get to the triplet state is by intersystem crossing,
> which is mostly based on spin-orbit coupling and depends a lot on the
> specific molecule and environment. But perhaps some FCS people have
> measured triplet population ratios after 2P excitation.
>
> I might be terribly wrong, my spectroscopy days were quite a while back,
> as was my QM course. Probably anyone else has some more input on this.
>
> Christian
>
> Am Dienstag, den 02.11.2010, 11:48 -0600 schrieb Craig Brideau:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Thanks for your insight, Christian.  So what it boils down to is that for
> > single photon interactions the primary method of damage is intrasystem
> > crossing into triplet states.  So the solution is to allow time for these
> > states to relax.  Conversely, for two photon the primary damage mechanism
> > are unwanted 3rd order or higher effects so lowering peak energy is the
> way
> > to go.  I'm just curious how much variability there is depending on your
> > sample.  Would triplet state excitation also be a factor at all in 2p, or
> > are the photon energies really low enough?  After all, couldn't 2p
> > interactions excite to triplet states as well?  Or is there just not
> enough
> > energy to cause crossing?
> >
> > Craig
> >
> >
> > On Tue, Nov 2, 2010 at 2:15 AM, Christian Schumann <
> > [hidden email]> wrote:
> >
> > > *****
> > > 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 <[hidden email]> 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: [phone]
> > > > > Email: [hidden email]
> > > > > http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng> <
> http://bme.pku.edu.cn/%7Exipeng> <
> > > http://bme.pku.edu.cn/%7Exipeng>
> > > > >
> > > > > On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <
> > > [hidden email]>
> > > > > 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: [phone]
> > > Telefax: [phone]
> > > E-Mail: [hidden email]
> > > 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
> > >
> ------------------------------------------------------------------------
> > >
> --
> Dr. Christian Schumann
> INM
> Leibniz-Institut für Neue Materialien gGmbH
> Campus D2 2
> 66123 Saarbrücken
>
> Telefon: [phone]
> Telefax: [phone]
> E-Mail: [hidden email]
> 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
> ------------------------------------------------------------------------
>

[Christian Schumann]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The "molecular memory" of the excitation process is lost after elaxation
from the Franck-Condon region, which happens within 50-200 fs. After
that, the molecular environment has the most influence of the reaction
route, and this is your best guess if you want to influence triplet
population (see  ie. Vogelsang et al., Angew Chem Int Ed, 47(29):5465).

Of course people have devised clever pulse shaping experiments to
prepare coherent wavepackets on the S1 surface to influence the course
of photochemical reactions. But although it's not impossible, I think it
would be extremly demanding to implement this in a confocal microscope,
where you have a lot of optics in your beam path as compared to
spectroscopy experiments. Also, optimization of the pulse shapes mostly
depends on feedback of the measurement of a reaction product and genetic
algorithms, which would also have to be implemented in the microscope.

Speaking of the spin of the photon: This is already required to fulfill
the selection rules for molecular excitation.

But it seems that this is getting really academic now ...

Christian

Am Dienstag, den 02.11.2010, 12:13 -0600 schrieb Craig Brideau:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Here's a question: Would the spin of the incident photons influence
> intersystem crossing?  As I understand it, crossing requires angular
> momentum changes.  If the incident photon possesses + or - spin would it
> make a difference?
>
> Craig
>
>
> On Tue, Nov 2, 2010 at 12:06 PM, Christian Schumann <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Craig,
> >
> > I don't know if there is much data out there on the 2P excitation cross
> > sections of triplet states, but I'd guess that these are probably not
> > really high.
> > Excitation directly to the triplet state is quantum mechanically not
> > allowed (at least for one-photon transitions) due to the unequal spin
> > multiplicities, so the cross section is practically non-existent. I'd
> > intuitively say that it's even lower for a 2P transition.
> > The only way to get to the triplet state is by intersystem crossing,
> > which is mostly based on spin-orbit coupling and depends a lot on the
> > specific molecule and environment. But perhaps some FCS people have
> > measured triplet population ratios after 2P excitation.
> >
> > I might be terribly wrong, my spectroscopy days were quite a while back,
> > as was my QM course. Probably anyone else has some more input on this.
> >
> > Christian
> >
> > Am Dienstag, den 02.11.2010, 11:48 -0600 schrieb Craig Brideau:
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Thanks for your insight, Christian.  So what it boils down to is that for
> > > single photon interactions the primary method of damage is intrasystem
> > > crossing into triplet states.  So the solution is to allow time for these
> > > states to relax.  Conversely, for two photon the primary damage mechanism
> > > are unwanted 3rd order or higher effects so lowering peak energy is the
> > way
> > > to go.  I'm just curious how much variability there is depending on your
> > > sample.  Would triplet state excitation also be a factor at all in 2p, or
> > > are the photon energies really low enough?  After all, couldn't 2p
> > > interactions excite to triplet states as well?  Or is there just not
> > enough
> > > energy to cause crossing?
> > >
> > > Craig
> > >
> > >
> > > On Tue, Nov 2, 2010 at 2:15 AM, Christian Schumann <
> > > [hidden email]> wrote:
> > >
> > > > *****
> > > > 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 <[hidden email]> 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: [phone]
> > > > > > Email: [hidden email]
> > > > > > http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng> <
> > http://bme.pku.edu.cn/%7Exipeng> <
> > > > http://bme.pku.edu.cn/%7Exipeng>
> > > > > >
> > > > > > On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <
> > > > [hidden email]>
> > > > > > 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: [phone]
> > > > Telefax: [phone]
> > > > E-Mail: [hidden email]
> > > > 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
> > > >
> > ------------------------------------------------------------------------
> > > >
> > --
> > Dr. Christian Schumann
> > INM
> > Leibniz-Institut für Neue Materialien gGmbH
> > Campus D2 2
> > 66123 Saarbrücken
> >
> > Telefon: [phone]
> > Telefax: [phone]
> > E-Mail: [hidden email]
> > 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
> > ------------------------------------------------------------------------
> >

--
Dr. Christian Schumann
INM
Leibniz-Institut für Neue Materialien gGmbH
Campus D2 2
66123 Saarbrücken

Telefon: [phone]
Telefax: [phone]
E-Mail: [hidden email]
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
------------------------------------------------------------------------