Pulse compression and in vivo imaging

[Stéphane Pagès]

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Hi everybody,
I am planning to image fluorescent neurons in vivo approximately  200 um
below the pia with a standard Ti:Sa laser.
I wonder if there is a clear advantage to use pulse compression to
compensate for dispersion of pulses due to tissue.
I understand theoretical arguments in favor of pulse compression.
However from an experimental point of view, are there some people here in
the list that have experienced some gain (in lowering the intensity of the
exciting beam for example).
Any comments would be greatly appreciated.
Thanks a lot
Stephane

[Craig Brideau]

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If scattering is the issue then adaptive optics will be more advantageous
than dispersion control.  The adaptive optics will help compensate somewhat
for the scattering and aberrations induced by the tissue.  To get good 2P
imaging you need a good focal spot more-so than you need a perfectly
transform limited pulse.  Adaptive optics will help keep your focus together
as you try to image deeply.  That said, dispersion compensation will help
somewhat so if you already have the necessary equipment then try it.

Craig



On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everybody,
> I am planning to image fluorescent neurons in vivo approximately  200 um
> below the pia with a standard Ti:Sa laser.
> I wonder if there is a clear advantage to use pulse compression to
> compensate for dispersion of pulses due to tissue.
> I understand theoretical arguments in favor of pulse compression.
> However from an experimental point of view, are there some people here in
> the list that have experienced some gain (in lowering the intensity of the
> exciting beam for example).
> Any comments would be greatly appreciated.
> Thanks a lot
> Stephane
>

[Stéphane Pagès]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks Craig,
Are you aware of some commercial optical adaptive systems  easily
implementable on a 2-P microscope ?
or is the technology still in development in labs ?



2011/7/7 Craig Brideau <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If scattering is the issue then adaptive optics will be more advantageous
> than dispersion control.  The adaptive optics will help compensate somewhat
> for the scattering and aberrations induced by the tissue.  To get good 2P
> imaging you need a good focal spot more-so than you need a perfectly
> transform limited pulse.  Adaptive optics will help keep your focus
> together
> as you try to image deeply.  That said, dispersion compensation will help
> somewhat so if you already have the necessary equipment then try it.
>
> Craig
>
>
>
> On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi everybody,
> > I am planning to image fluorescent neurons in vivo approximately  200 um
> > below the pia with a standard Ti:Sa laser.
> > I wonder if there is a clear advantage to use pulse compression to
> > compensate for dispersion of pulses due to tissue.
> > I understand theoretical arguments in favor of pulse compression.
> > However from an experimental point of view, are there some people here in
> > the list that have experienced some gain (in lowering the intensity of
> the
> > exciting beam for example).
> > Any comments would be greatly appreciated.
> > Thanks a lot
> > Stephane
> >
>

[Philippe clemenceau]

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Hello Stephane,

My company, Imagine Optic, manufactures high performance Adaptive Optics
loops and we have lots of experience and very good results in microscopy,
especially non-linear microscopy and super resolution.

You can go to
http://www.imagine-optic.com/iop_applications_adaptive-optics_microscopy-lif
e-sciences_en.php to see some images.

To avoid too commercial language on the list, I will send you a private
email.

Kind regards,

Philippe Clémenceau, Division Manager

Imagine Optic Inc./Axiom Optics
Ph:[phone]
Cell: + 1 [phone]
1 Broadway, 14th floor
Fax: [phone]
Cambridge,  MA 02142
www.imagine-optic.com

Scientific Imaging , Laser Characterization & Laser based NDT

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Stéphane Pagès
Sent: Friday, July 08, 2011 4:23 AM
To: [hidden email]
Subject: Re: Pulse compression and in vivo imaging

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Thanks Craig,
Are you aware of some commercial optical adaptive systems  easily
implementable on a 2-P microscope ?
or is the technology still in development in labs ?



2011/7/7 Craig Brideau <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If scattering is the issue then adaptive optics will be more advantageous
> than dispersion control.  The adaptive optics will help compensate
somewhat

> for the scattering and aberrations induced by the tissue.  To get good 2P
> imaging you need a good focal spot more-so than you need a perfectly
> transform limited pulse.  Adaptive optics will help keep your focus
> together
> as you try to image deeply.  That said, dispersion compensation will help
> somewhat so if you already have the necessary equipment then try it.
>
> Craig
>
>
>
> On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi everybody,
> > I am planning to image fluorescent neurons in vivo approximately  200 um
> > below the pia with a standard Ti:Sa laser.
> > I wonder if there is a clear advantage to use pulse compression to
> > compensate for dispersion of pulses due to tissue.
> > I understand theoretical arguments in favor of pulse compression.
> > However from an experimental point of view, are there some people here

in
> > the list that have experienced some gain (in lowering the intensity of
> the
> > exciting beam for example).
> > Any comments would be greatly appreciated.
> > Thanks a lot
> > Stephane
> >
>

-----
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Analyse effectuée par AVG - www.avg.fr
Version: 10.0.1388 / Base de données virale: 1516/3750 - Date: 07/07/2011

[Craig Brideau]

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Heh.  That was quick for the commercial response. @:-) Some of the adaptive
optics companies, like these guys, have drop-in systems.  I haven't
personally used one but from what I've seen and heard they are relatively
easy to add to existing setups.  It is interesting how quickly the
commercial implementation of AO has progressed. It has gone from lab-only to
fast add-on within a couple years.  I guess everyone is hoping that adaptive
optics can do for microscopy what it has done for astronomy.

Craig


On Fri, Jul 8, 2011 at 8:08 AM, Philippe Clemenceau <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Stephane,
>
> My company, Imagine Optic, manufactures high performance Adaptive Optics
> loops and we have lots of experience and very good results in microscopy,
> especially non-linear microscopy and super resolution.
>
> You can go to
>
> http://www.imagine-optic.com/iop_applications_adaptive-optics_microscopy-lif
> e-sciences_en.php to see some images.
>
> To avoid too commercial language on the list, I will send you a private
> email.
>
> Kind regards,
>
> Philippe Clémenceau, Division Manager
>
> Imagine Optic Inc./Axiom Optics
> Ph:[phone]
> Cell: + 1 [phone]
> 1 Broadway, 14th floor
> Fax: [phone]
> Cambridge,  MA 02142
> www.imagine-optic.com
>
> Scientific Imaging , Laser Characterization & Laser based NDT
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On
> Behalf Of Stéphane Pagès
> Sent: Friday, July 08, 2011 4:23 AM
> To: [hidden email]
> Subject: Re: Pulse compression and in vivo imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks Craig,
> Are you aware of some commercial optical adaptive systems  easily
> implementable on a 2-P microscope ?
> or is the technology still in development in labs ?
>
>
>
> 2011/7/7 Craig Brideau <[hidden email]>
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > If scattering is the issue then adaptive optics will be more advantageous
> > than dispersion control.  The adaptive optics will help compensate
> somewhat
> > for the scattering and aberrations induced by the tissue.  To get good 2P
> > imaging you need a good focal spot more-so than you need a perfectly
> > transform limited pulse.  Adaptive optics will help keep your focus
> > together
> > as you try to image deeply.  That said, dispersion compensation will help
> > somewhat so if you already have the necessary equipment then try it.
> >
> > Craig
> >
> >
> >
> > On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Hi everybody,
> > > I am planning to image fluorescent neurons in vivo approximately  200
> um
> > > below the pia with a standard Ti:Sa laser.
> > > I wonder if there is a clear advantage to use pulse compression to
> > > compensate for dispersion of pulses due to tissue.
> > > I understand theoretical arguments in favor of pulse compression.
> > > However from an experimental point of view, are there some people here
> in
> > > the list that have experienced some gain (in lowering the intensity of
> > the
> > > exciting beam for example).
> > > Any comments would be greatly appreciated.
> > > Thanks a lot
> > > Stephane
> > >
> >
>
> -----
> Aucun virus trouvé dans ce message.
> Analyse effectuée par AVG - www.avg.fr
> Version: 10.0.1388 / Base de données virale: 1516/3750 - Date: 07/07/2011
>

[Stanislav Vitha]

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Also, it depends on the pulse width.
the shorter the pulse, the more you may need the dispersion control as you go
deeper in the sample.
On our system with 10 fs pulses, we really cannot live without pre-chirp
(dispersion control). Your standard oscillator (~100-fs pulses?) is much more
forgiving.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University


On Thu, 7 Jul 2011 13:20:02 -0600, 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
>*****
>
>If scattering is the issue then adaptive optics will be more advantageous
>than dispersion control.  The adaptive optics will help compensate somewhat
>for the scattering and aberrations induced by the tissue.  To get good 2P
>imaging you need a good focal spot more-so than you need a perfectly
>transform limited pulse.  Adaptive optics will help keep your focus together
>as you try to image deeply.  That said, dispersion compensation will help
>somewhat so if you already have the necessary equipment then try it.
>
>Craig
>
>
>
>On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
>[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everybody,
>> I am planning to image fluorescent neurons in vivo approximately  200 um
>> below the pia with a standard Ti:Sa laser.
>> I wonder if there is a clear advantage to use pulse compression to
>> compensate for dispersion of pulses due to tissue.
>> I understand theoretical arguments in favor of pulse compression.
>> However from an experimental point of view, are there some people here

in
>> the list that have experienced some gain (in lowering the intensity of the
>> exciting beam for example).
>> Any comments would be greatly appreciated.
>> Thanks a lot
>> Stephane
>>

[Craig Brideau]

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

Actually the Chameleon was designed for 140fs pulses for this reason.  For a
'typical' microscope system, transform-limited 140 fs gives you minimum
dispersion.  On the other hand, if you have a pulse compressor you want to
start with as short a pulse as possible as the compressor can keep
dispersion in check for you.
If I might ask, Stan, what do you use for a compressor on your 10fs laser?

Craig


On Fri, Jul 8, 2011 at 1:57 PM, Stanislav Vitha <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Also, it depends on the pulse width.
> the shorter the pulse, the more you may need the dispersion control as you
> go
> deeper in the sample.
> On our system with 10 fs pulses, we really cannot live without pre-chirp
> (dispersion control). Your standard oscillator (~100-fs pulses?) is much
> more
> forgiving.
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
> On Thu, 7 Jul 2011 13:20:02 -0600, 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
> >*****
> >
> >If scattering is the issue then adaptive optics will be more advantageous
> >than dispersion control.  The adaptive optics will help compensate
> somewhat
> >for the scattering and aberrations induced by the tissue.  To get good 2P
> >imaging you need a good focal spot more-so than you need a perfectly
> >transform limited pulse.  Adaptive optics will help keep your focus
> together
> >as you try to image deeply.  That said, dispersion compensation will help
> >somewhat so if you already have the necessary equipment then try it.
> >
> >Craig
> >
> >
> >
> >On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
> >[hidden email]> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hi everybody,
> >> I am planning to image fluorescent neurons in vivo approximately  200 um
> >> below the pia with a standard Ti:Sa laser.
> >> I wonder if there is a clear advantage to use pulse compression to
> >> compensate for dispersion of pulses due to tissue.
> >> I understand theoretical arguments in favor of pulse compression.
> >> However from an experimental point of view, are there some people here
> in
> >> the list that have experienced some gain (in lowering the intensity of
> the
> >> exciting beam for example).
> >> Any comments would be greatly appreciated.
> >> Thanks a lot
> >> Stephane
> >>
>

[James Pawley]

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

Just a comment. Numerous studies on phototoxicity
have shown that, in both single- and 2-photon
microscopy, damage is (at least) proportional to
the number of molecular excitations. If this
holds, then if shorter pulses provide more
(non-descanned) signal, it should also produce
more photodamage.

In addition, Dave Piston often made two points:
that damage/excitation was often more severe with
2-photon than single-photon excitation, and that,
(depending on the wavelength) the shorter,
higher-peak-power pulses that increase 2-photon
signal may also increase 3-photon excitation of
natural fluorophors in the cell.

Have any of you noticed more photodamage when
using shorter pulses? (Photodamage can cover a
lot of effects from exploding cells to cells that
should divide but fail to do so. Any change would
be of interest to me.)

Finally, more intense pulses means that the
"threshold" for 2-photon excitation will be
reached farther above and below the expected
plane of focus than would be the case with
longer, less intense pulses. i.e., at least some
of the extra signal seen with shorter pulses may
be the result of the PSF being larger in x,y and
z, meaning that you excite more dye molecules.
(As one moves above or below the focus plane, the
hour-glass PSF becomes wider as well as taller.)

Has anyone seen a change in resolution when using shorter pulses?

Cheers,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Also, it depends on the pulse width.
>the shorter the pulse, the more you may need the dispersion control as you go
>deeper in the sample.
>On our system with 10 fs pulses, we really cannot live without pre-chirp
>(dispersion control). Your standard oscillator (~100-fs pulses?) is much more
>forgiving.
>
>Stan Vitha
>Microscopy and Imaging Center
>Texas A&M University
>
>
>On Thu, 7 Jul 2011 13:20:02 -0600, 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
>>*****
>>
>>If scattering is the issue then adaptive optics will be more advantageous
>>than dispersion control.  The adaptive optics will help compensate somewhat
>>for the scattering and aberrations induced by the tissue.  To get good 2P
>>imaging you need a good focal spot more-so than you need a perfectly
>>transform limited pulse.  Adaptive optics will help keep your focus together
>>as you try to image deeply.  That said, dispersion compensation will help
>>somewhat so if you already have the necessary equipment then try it.
>>
>>Craig
>>
>>
>>
>>On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
>>[hidden email]> wrote:
>>
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>  *****
>>>
>>>  Hi everybody,
>>>  I am planning to image fluorescent neurons in vivo approximately  200 um
>>>  below the pia with a standard Ti:Sa laser.
>>>  I wonder if there is a clear advantage to use pulse compression to
>>>  compensate for dispersion of pulses due to tissue.
>>>  I understand theoretical arguments in favor of pulse compression.
>>>  However from an experimental point of view, are there some people here
>in
>>>  the list that have experienced some gain (in lowering the intensity of the
>>>  exciting beam for example).
>>>  Any comments would be greatly appreciated.
>>>  Thanks a lot
>>>  Stephane
>>>

--
Jim Pawley (Summer address) c/o Postmaster,
Egmont, BC, Canada, V0N-1N0 604-883-2095,
[hidden email]

[Craig Brideau]

*****
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*****

We have a home-built pulse compressor that lets us get the pulses from our
Tsunami nearly transform limited at the sample.  They are about 60fs when
transform limited.  The first thing we noticed was a significant increase in
tissue destruction when imaging live tissue, even with the average laser
power turned down to minimum.  We did get an order of magnitude increase is
signal, but the photodamage effects made things impractical.  After some
empirical adjusting, we found that around 150-200fs gave reasonable signal
at reasonable powers with minimum tissue destruction.  Our samples were
being perfused since they were live though, so there was plenty of oxygen in
the environment for free radical generation.  Your mileage may vary by
sample.  As for resolution, no particular improvement was noticed.

Craig


On Fri, Jul 8, 2011 at 7:04 PM, James Pawley <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Hi all,
>
> Just a comment. Numerous studies on phototoxicity have shown that, in both
> single- and 2-photon microscopy, damage is (at least) proportional to the
> number of molecular excitations. If this holds, then if shorter pulses
> provide more (non-descanned) signal, it should also produce more
> photodamage.
>
> In addition, Dave Piston often made two points: that damage/excitation was
> often more severe with 2-photon than single-photon excitation, and that,
> (depending on the wavelength) the shorter, higher-peak-power pulses that
> increase 2-photon signal may also increase 3-photon excitation of natural
> fluorophors in the cell.
>
> Have any of you noticed more photodamage when using shorter pulses?
> (Photodamage can cover a lot of effects from exploding cells to cells that
> should divide but fail to do so. Any change would be of interest to me.)
>
> Finally, more intense pulses means that the "threshold" for 2-photon
> excitation will be reached farther above and below the expected plane of
> focus than would be the case with longer, less intense pulses. i.e., at
> least some of the extra signal seen with shorter pulses may be the result of
> the PSF being larger in x,y and z, meaning that you excite more dye
> molecules. (As one moves above or below the focus plane, the hour-glass PSF
> becomes wider as well as taller.)
>
> Has anyone seen a change in resolution when using shorter pulses?
>
> Cheers,
>
> Jim Pawley
>
>
>
>  *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Also, it depends on the pulse width.
>> the shorter the pulse, the more you may need the dispersion control as you
>> go
>> deeper in the sample.
>> On our system with 10 fs pulses, we really cannot live without pre-chirp
>> (dispersion control). Your standard oscillator (~100-fs pulses?) is much
>> more
>> forgiving.
>>
>> Stan Vitha
>> Microscopy and Imaging Center
>> Texas A&M University
>>
>>
>> On Thu, 7 Jul 2011 13:20:02 -0600, 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<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>> *****
>>>
>>> If scattering is the issue then adaptive optics will be more advantageous
>>> than dispersion control.  The adaptive optics will help compensate
>>> somewhat
>>> for the scattering and aberrations induced by the tissue.  To get good 2P
>>> imaging you need a good focal spot more-so than you need a perfectly
>>> transform limited pulse.  Adaptive optics will help keep your focus
>>> together
>>> as you try to image deeply.  That said, dispersion compensation will help
>>> somewhat so if you already have the necessary equipment then try it.
>>>
>>> Craig
>>>
>>>
>>>
>>> On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
>>> [hidden email].**ca <[hidden email]>>
>>> wrote:
>>>
>>>   *****
>>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>>  http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>>>  *****
>>>>
>>>>  Hi everybody,
>>>>  I am planning to image fluorescent neurons in vivo approximately  200
>>>> um
>>>>  below the pia with a standard Ti:Sa laser.
>>>>  I wonder if there is a clear advantage to use pulse compression to
>>>>  compensate for dispersion of pulses due to tissue.
>>>>  I understand theoretical arguments in favor of pulse compression.
>>>>  However from an experimental point of view, are there some people here
>>>>
>>> in
>>
>>>  the list that have experienced some gain (in lowering the intensity of
>>>> the
>>>>  exciting beam for example).
>>>>  Any comments would be greatly appreciated.
>>>>  Thanks a lot
>>>>  Stephane
>>>>
>>>>
>
> --
> Jim Pawley (Summer address) c/o Postmaster, Egmont, BC, Canada, V0N-1N0
> 604-883-2095, [hidden email]
>

[Guy Cox-2]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

There are a lot of points here.  (Actually I suppose it should be 'There is a lot ....')

Firstly, shorter pulses will not make the psf larger.  The psf depends on the wavelength, nothing else (and is, in principle, infinite ...)  The only thing that will make it appear to spread is if you are saturating the fluorescence at the centre of the psf, and this will apply irrespective of whether you are doing 2P or regular confocal.  There are all sorts of reasons why saturation is a bad thing which we need not go into here.  

If you shorten the pulse while keeping the power the same you will increase all 2 and 3 photon processes, both damaging and not.  Since 2P follows a square law and 3P a cube law you will change the relative proportions, as Jim says. If you shorten the pulses and keep the peak intensity the same you will reduce overall power which at least will reduce heating of the sample so there should be some benefit, without affecting multi-photon processes.  I suspect that what most people do is something in between.

Why is 2P more damaging?  Arguably it's not - Vadim Dedov and I were able to measure mitochondrial membrane potential in nerve cells with JC1 using 2-photon excitation while equivalent single-photon excitation killed the cells and we couldn't measure anything.  There are lots of other examples in the literature.  What is true is that 2P can cause different sorts of damage.  The most extreme is breakdown caused by the electric field, which appears as bright flashes as you scan and 'craters' thereafter.  If you increase the peak electric field you will naturally increase this damage.

Another point is more subtle.  Chemical selection rules state that in a symmetrical molecule, 2P excitation must occur to a different state than 1P.  This means you will not excite the S1 state, and hence you have an enhanced rate of inter-system crossing into a triplet state.  This is a very noticeable with fluorescein, since it is symmetrical.  There are lots of published spectra out there now - if a fluorochrome shows very different 1P and 2P spectra you'd do best to avoid it.

Finally, when we compress pulses we may not get what we think we are getting.  Chirping gives a pulse a strange shape, which we hope will even out to a normal pulse after passing through our optics.  If in fact we excite with a chirped pulse then the peak intensity may be much high higher than we'd calculate from the nominal pulse length and average power.  

                                                Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Saturday, 9 July 2011 11:05 AM
To: [hidden email]
Subject: Re: Pulse compression and in vivo imaging

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

Hi all,

Just a comment. Numerous studies on phototoxicity
have shown that, in both single- and 2-photon
microscopy, damage is (at least) proportional to
the number of molecular excitations. If this
holds, then if shorter pulses provide more
(non-descanned) signal, it should also produce
more photodamage.

In addition, Dave Piston often made two points:
that damage/excitation was often more severe with
2-photon than single-photon excitation, and that,
(depending on the wavelength) the shorter,
higher-peak-power pulses that increase 2-photon
signal may also increase 3-photon excitation of
natural fluorophors in the cell.

Have any of you noticed more photodamage when
using shorter pulses? (Photodamage can cover a
lot of effects from exploding cells to cells that
should divide but fail to do so. Any change would
be of interest to me.)

Finally, more intense pulses means that the
"threshold" for 2-photon excitation will be
reached farther above and below the expected
plane of focus than would be the case with
longer, less intense pulses. i.e., at least some
of the extra signal seen with shorter pulses may
be the result of the PSF being larger in x,y and
z, meaning that you excite more dye molecules.
(As one moves above or below the focus plane, the
hour-glass PSF becomes wider as well as taller.)

Has anyone seen a change in resolution when using shorter pulses?

Cheers,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Also, it depends on the pulse width.
>the shorter the pulse, the more you may need the dispersion control as you go
>deeper in the sample.
>On our system with 10 fs pulses, we really cannot live without pre-chirp
>(dispersion control). Your standard oscillator (~100-fs pulses?) is much more
>forgiving.
>
>Stan Vitha
>Microscopy and Imaging Center
>Texas A&M University
>
>
>On Thu, 7 Jul 2011 13:20:02 -0600, 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
>>*****
>>
>>If scattering is the issue then adaptive optics will be more advantageous
>>than dispersion control.  The adaptive optics will help compensate somewhat
>>for the scattering and aberrations induced by the tissue.  To get good 2P
>>imaging you need a good focal spot more-so than you need a perfectly
>>transform limited pulse.  Adaptive optics will help keep your focus together
>>as you try to image deeply.  That said, dispersion compensation will help
>>somewhat so if you already have the necessary equipment then try it.
>>
>>Craig
>>
>>
>>
>>On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
>>[hidden email]> wrote:
>>
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>  *****
>>>
>>>  Hi everybody,
>>>  I am planning to image fluorescent neurons in vivo approximately  200 um
>>>  below the pia with a standard Ti:Sa laser.
>>>  I wonder if there is a clear advantage to use pulse compression to
>>>  compensate for dispersion of pulses due to tissue.
>>>  I understand theoretical arguments in favor of pulse compression.
>>>  However from an experimental point of view, are there some people here
>in
>>>  the list that have experienced some gain (in lowering the intensity of the
>>>  exciting beam for example).
>>>  Any comments would be greatly appreciated.
>>>  Thanks a lot
>>>  Stephane
>>>

--
Jim Pawley (Summer address) c/o Postmaster,
Egmont, BC, Canada, V0N-1N0 604-883-2095,
[hidden email]

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1388 / Virus Database: 1516/3751 - Release Date: 07/08/11

[Stanislav Vitha]

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

Craig,
We use a pair of chirp mirrors, typically set-up with 7 or 8 pairs of bounces  
(depends on the objective used). We slightly over-compensate and have
several mm of BK7 glass in the laser path, that we can take out as needed for
deep imaging.
The slight downside of the current set up is that changing the number of
bounces on the chirp mirrors requires laser re-alignment.  


Stan
 

On Fri, 8 Jul 2011 14:32:40 -0600, Craig Brideau <[hidden email]>
wrote:

>*****
>
>Actually the Chameleon was designed for 140fs pulses for this reason.  For a
>'typical' microscope system, transform-limited 140 fs gives you minimum
>dispersion.  On the other hand, if you have a pulse compressor you want to
>start with as short a pulse as possible as the compressor can keep
>dispersion in check for you.
>If I might ask, Stan, what do you use for a compressor on your 10fs laser?
>
>Craig
>
>
>On Fri, Jul 8, 2011 at 1:57 PM, Stanislav Vitha <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Also, it depends on the pulse width.
>> the shorter the pulse, the more you may need the dispersion control as you
>> go
>> deeper in the sample.
>> On our system with 10 fs pulses, we really cannot live without pre-chirp
>> (dispersion control). Your standard oscillator (~100-fs pulses?) is much
>> more
>> forgiving.
>>
>> Stan Vitha
>> Microscopy and Imaging Center
>> Texas A&M University
>>

[Craig Brideau]

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

I feel your pain!  I have a pair of prisms in my compressor.  Whenever I
change my Ti:Saph's wavelength I have to realign the prisms.  The automated
systems like the Deepsee and Vision  do this for you, but they are
expensive.  One trick I am currently working on is overcompensating with
chirped mirrors, then using glass wedges to bring the pulse back into
optimum.  If I put the wedges on a translation stage it is a simple matter
to slide a wedge in and out of the beam path.  It would only be a single
adjustment, and I can mark the micrometer which moves the wedge for
different wavelengths.  I am going to have a set of wedges custom-designed
for Ti:Saph lasers.  I had great success with this for my prism compressor.
 I got a company to cut and anti-reflection coat them to my design; I plan
on having the same thing done for the wedges.  Since we're talking about it,
would anyone be interested in a positive-dispersion-control pair of glass
wedges designed for Ti:Saph lasers?  The more the company produces for me in
a batch the cheaper they are individually.  If anyone wants to get on-board
give me an email; prices will probably be under $1000 for two wedges with AR
coatings and angles optimized for Ti:Saph lasers.  You put the two wedges
into a beam and move one or both back and forth.  This does not deviate the
beam and allows you to put controlled amounts of glass (I'm probably going
with fused silica) into the beam.  This lets you put controlled amounts of
positive dispersion on the beam to counteract an excess of negative from
your chirped mirrors or whatever you are using.

Craig


On Sat, Jul 9, 2011 at 1:26 PM, Stanislav Vitha <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Craig,
> We use a pair of chirp mirrors, typically set-up with 7 or 8 pairs of
> bounces
> (depends on the objective used). We slightly over-compensate and have
> several mm of BK7 glass in the laser path, that we can take out as needed
> for
> deep imaging.
> The slight downside of the current set up is that changing the number of
> bounces on the chirp mirrors requires laser re-alignment.
>
>
> Stan
>
>
> On Fri, 8 Jul 2011 14:32:40 -0600, Craig Brideau <[hidden email]>
> wrote:
>
> >*****
> >
> >Actually the Chameleon was designed for 140fs pulses for this reason.  For
> a
> >'typical' microscope system, transform-limited 140 fs gives you minimum
> >dispersion.  On the other hand, if you have a pulse compressor you want to
> >start with as short a pulse as possible as the compressor can keep
> >dispersion in check for you.
> >If I might ask, Stan, what do you use for a compressor on your 10fs laser?
> >
> >Craig
> >
> >
> >On Fri, Jul 8, 2011 at 1:57 PM, Stanislav Vitha <[hidden email]>
> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Also, it depends on the pulse width.
> >> the shorter the pulse, the more you may need the dispersion control as
> you
> >> go
> >> deeper in the sample.
> >> On our system with 10 fs pulses, we really cannot live without pre-chirp
> >> (dispersion control). Your standard oscillator (~100-fs pulses?) is much
> >> more
> >> forgiving.
> >>
> >> Stan Vitha
> >> Microscopy and Imaging Center
> >> Texas A&M University
> >>
>

[Andreas Bruckbauer]

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

Is there a way to estimate how much of the damage comes from photochemistry and how much is heating? Is it possibe to measure temperature using fluorescence to determine if the sample is locally heated?

best wishes

Andreas



 

 


 

 

-----Original Message-----
From: Guy Cox <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sat, 9 Jul 2011 9:16
Subject: Re: Pulse compression and in vivo imaging


*****

To join, leave or search the confocal microscopy listserv, go to:

http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

*****



There are a lot of points here.  (Actually I suppose it should be 'There is a

lot ....')



Firstly, shorter pulses will not make the psf larger.  The psf depends on the

wavelength, nothing else (and is, in principle, infinite ...)  The only thing

that will make it appear to spread is if you are saturating the fluorescence at

the centre of the psf, and this will apply irrespective of whether you are doing

2P or regular confocal.  There are all sorts of reasons why saturation is a bad

thing which we need not go into here.  



If you shorten the pulse while keeping the power the same you will increase all

2 and 3 photon processes, both damaging and not.  Since 2P follows a square law

and 3P a cube law you will change the relative proportions, as Jim says. If you

shorten the pulses and keep the peak intensity the same you will reduce overall

power which at least will reduce heating of the sample so there should be some

benefit, without affecting multi-photon processes.  I suspect that what most

people do is something in between.



Why is 2P more damaging?  Arguably it's not - Vadim Dedov and I were able to

measure mitochondrial membrane potential in nerve cells with JC1 using 2-photon

excitation while equivalent single-photon excitation killed the cells and we

couldn't measure anything.  There are lots of other examples in the literature.  

What is true is that 2P can cause different sorts of damage.  The most extreme

is breakdown caused by the electric field, which appears as bright flashes as

you scan and 'craters' thereafter.  If you increase the peak electric field you

will naturally increase this damage.



Another point is more subtle.  Chemical selection rules state that in a

symmetrical molecule, 2P excitation must occur to a different state than 1P.  

This means you will not excite the S1 state, and hence you have an enhanced rate

of inter-system crossing into a triplet state.  This is a very noticeable with

fluorescein, since it is symmetrical.  There are lots of published spectra out

there now - if a fluorochrome shows very different 1P and 2P spectra you'd do

best to avoid it.



Finally, when we compress pulses we may not get what we think we are getting.  

Chirping gives a pulse a strange shape, which we hope will even out to a normal

pulse after passing through our optics.  If in fact we excite with a chirped

pulse then the peak intensity may be much high higher than we'd calculate from

the nominal pulse length and average power.  



                                                Guy





Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006



Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net

 



-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of James Pawley

Sent: Saturday, 9 July 2011 11:05 AM

To: [hidden email]

Subject: Re: Pulse compression and in vivo imaging



*****

To join, leave or search the confocal microscopy listserv, go to:

http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

*****



Hi all,



Just a comment. Numerous studies on phototoxicity

have shown that, in both single- and 2-photon

microscopy, damage is (at least) proportional to

the number of molecular excitations. If this

holds, then if shorter pulses provide more

(non-descanned) signal, it should also produce

more photodamage.



In addition, Dave Piston often made two points:

that damage/excitation was often more severe with

2-photon than single-photon excitation, and that,

(depending on the wavelength) the shorter,

higher-peak-power pulses that increase 2-photon

signal may also increase 3-photon excitation of

natural fluorophors in the cell.



Have any of you noticed more photodamage when

using shorter pulses? (Photodamage can cover a

lot of effects from exploding cells to cells that

should divide but fail to do so. Any change would

be of interest to me.)



Finally, more intense pulses means that the

"threshold" for 2-photon excitation will be

reached farther above and below the expected

plane of focus than would be the case with

longer, less intense pulses. i.e., at least some

of the extra signal seen with shorter pulses may

be the result of the PSF being larger in x,y and

z, meaning that you excite more dye molecules.

(As one moves above or below the focus plane, the

hour-glass PSF becomes wider as well as taller.)



Has anyone seen a change in resolution when using shorter pulses?



Cheers,



Jim Pawley





>*****

>To join, leave or search the confocal microscopy listserv, go to:

>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

>*****

>

>Also, it depends on the pulse width.

>the shorter the pulse, the more you may need the dispersion control as you go

>deeper in the sample.

>On our system with 10 fs pulses, we really cannot live without pre-chirp

>(dispersion control). Your standard oscillator (~100-fs pulses?) is much more

>forgiving.

>

>Stan Vitha

>Microscopy and Imaging Center

>Texas A&M University

>

>

>On Thu, 7 Jul 2011 13:20:02 -0600, 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

>>*****

>>

>>If scattering is the issue then adaptive optics will be more advantageous

>>than dispersion control.  The adaptive optics will help compensate somewhat

>>for the scattering and aberrations induced by the tissue.  To get good 2P

>>imaging you need a good focal spot more-so than you need a perfectly

>>transform limited pulse.  Adaptive optics will help keep your focus together

>>as you try to image deeply.  That said, dispersion compensation will help

>>somewhat so if you already have the necessary equipment then try it.

>>

>>Craig

>>

>>

>>

>>On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <

>>[hidden email]> wrote:

>>

>>>  *****

>>>  To join, leave or search the confocal microscopy listserv, go to:

>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

>>>  *****

>>>

>>>  Hi everybody,

>>>  I am planning to image fluorescent neurons in vivo approximately  200 um

>>>  below the pia with a standard Ti:Sa laser.

>>>  I wonder if there is a clear advantage to use pulse compression to

>>>  compensate for dispersion of pulses due to tissue.

>>>  I understand theoretical arguments in favor of pulse compression.

>>>  However from an experimental point of view, are there some people here

>in

>>>  the list that have experienced some gain (in lowering the intensity of the

>>>  exciting beam for example).

>>>  Any comments would be greatly appreciated.

>>>  Thanks a lot

>>>  Stephane

>>>





--

Jim Pawley (Summer address) c/o Postmaster,

Egmont, BC, Canada, V0N-1N0 604-883-2095,

[hidden email]



-----

No virus found in this message.

Checked by AVG - www.avg.com

Version: 10.0.1388 / Virus Database: 1516/3751 - Release Date: 07/08/11


 

[Craig Brideau]

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

Heating is going to be highly sample dependent.  Thermal effects are by
absorption of the energy.  Since most tissues are transparent to NIR they
don't tend to absorb much so you need significant wattage delivered to
generate heating.  That said, depending on your tissue your mileage may
vary.  For instance a perfused sample in solution will probably handle more
heating than a fixed thin section on a slide.  Non-linear effects tend not
to care about wavelength if the pulses are short enough.  Basically the
transient energy becomes so high that you get ionizing effects which is how
you get photo-chemical effects.  I'm sure wiser minds can give a more
elegant description of this, but that's the gist of it.

Craig

On Sat, Jul 9, 2011 at 2:37 PM, Andreas Bruckbauer <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Is there a way to estimate how much of the damage comes from photochemistry
> and how much is heating? Is it possibe to measure temperature using
> fluorescence to determine if the sample is locally heated?
>
> best wishes
>
> Andreas
>
>
>
>
>
>
>
>
>
>
>
>
> -----Original Message-----
> From: Guy Cox <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Sat, 9 Jul 2011 9:16
> Subject: Re: Pulse compression and in vivo imaging
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> There are a lot of points here.  (Actually I suppose it should be 'There is
> a
>
> lot ....')
>
>
>
> Firstly, shorter pulses will not make the psf larger.  The psf depends on
> the
>
> wavelength, nothing else (and is, in principle, infinite ...)  The only
> thing
>
> that will make it appear to spread is if you are saturating the
> fluorescence at
>
> the centre of the psf, and this will apply irrespective of whether you are
> doing
>
> 2P or regular confocal.  There are all sorts of reasons why saturation is a
> bad
>
> thing which we need not go into here.
>
>
>
> If you shorten the pulse while keeping the power the same you will increase
> all
>
> 2 and 3 photon processes, both damaging and not.  Since 2P follows a square
> law
>
> and 3P a cube law you will change the relative proportions, as Jim says. If
> you
>
> shorten the pulses and keep the peak intensity the same you will reduce
> overall
>
> power which at least will reduce heating of the sample so there should be
> some
>
> benefit, without affecting multi-photon processes.  I suspect that what
> most
>
> people do is something in between.
>
>
>
> Why is 2P more damaging?  Arguably it's not - Vadim Dedov and I were able
> to
>
> measure mitochondrial membrane potential in nerve cells with JC1 using
> 2-photon
>
> excitation while equivalent single-photon excitation killed the cells and
> we
>
> couldn't measure anything.  There are lots of other examples in the
> literature.
>
> What is true is that 2P can cause different sorts of damage.  The most
> extreme
>
> is breakdown caused by the electric field, which appears as bright flashes
> as
>
> you scan and 'craters' thereafter.  If you increase the peak electric field
> you
>
> will naturally increase this damage.
>
>
>
> Another point is more subtle.  Chemical selection rules state that in a
>
> symmetrical molecule, 2P excitation must occur to a different state than
> 1P.
>
> This means you will not excite the S1 state, and hence you have an enhanced
> rate
>
> of inter-system crossing into a triplet state.  This is a very noticeable
> with
>
> fluorescein, since it is symmetrical.  There are lots of published spectra
> out
>
> there now - if a fluorochrome shows very different 1P and 2P spectra you'd
> do
>
> best to avoid it.
>
>
>
> Finally, when we compress pulses we may not get what we think we are
> getting.
>
> Chirping gives a pulse a strange shape, which we hope will even out to a
> normal
>
> pulse after passing through our optics.  If in fact we excite with a
> chirped
>
> pulse then the peak intensity may be much high higher than we'd calculate
> from
>
> the nominal pulse length and average power.
>
>
>
>                                                Guy
>
>
>
>
>
> Optical Imaging Techniques in Cell Biology
>
> by Guy Cox    CRC Press / Taylor & Francis
>
>     http://www.guycox.com/optical.htm
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone [phone]     Fax [phone]
>
>             Mobile [phone]
>
> ______________________________________________
>
>      http://www.guycox.net
>
>
>
>
>
> -----Original Message-----
>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On
>
> Behalf Of James Pawley
>
> Sent: Saturday, 9 July 2011 11:05 AM
>
> To: [hidden email]
>
> Subject: Re: Pulse compression and in vivo imaging
>
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Hi all,
>
>
>
> Just a comment. Numerous studies on phototoxicity
>
> have shown that, in both single- and 2-photon
>
> microscopy, damage is (at least) proportional to
>
> the number of molecular excitations. If this
>
> holds, then if shorter pulses provide more
>
> (non-descanned) signal, it should also produce
>
> more photodamage.
>
>
>
> In addition, Dave Piston often made two points:
>
> that damage/excitation was often more severe with
>
> 2-photon than single-photon excitation, and that,
>
> (depending on the wavelength) the shorter,
>
> higher-peak-power pulses that increase 2-photon
>
> signal may also increase 3-photon excitation of
>
> natural fluorophors in the cell.
>
>
>
> Have any of you noticed more photodamage when
>
> using shorter pulses? (Photodamage can cover a
>
> lot of effects from exploding cells to cells that
>
> should divide but fail to do so. Any change would
>
> be of interest to me.)
>
>
>
> Finally, more intense pulses means that the
>
> "threshold" for 2-photon excitation will be
>
> reached farther above and below the expected
>
> plane of focus than would be the case with
>
> longer, less intense pulses. i.e., at least some
>
> of the extra signal seen with shorter pulses may
>
> be the result of the PSF being larger in x,y and
>
> z, meaning that you excite more dye molecules.
>
> (As one moves above or below the focus plane, the
>
> hour-glass PSF becomes wider as well as taller.)
>
>
>
> Has anyone seen a change in resolution when using shorter pulses?
>
>
>
> Cheers,
>
>
>
> Jim Pawley
>
>
>
>
>
> >*****
>
> >To join, leave or search the confocal microscopy listserv, go to:
>
> >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> >*****
>
> >
>
> >Also, it depends on the pulse width.
>
> >the shorter the pulse, the more you may need the dispersion control as you
> go
>
> >deeper in the sample.
>
> >On our system with 10 fs pulses, we really cannot live without pre-chirp
>
> >(dispersion control). Your standard oscillator (~100-fs pulses?) is much
> more
>
> >forgiving.
>
> >
>
> >Stan Vitha
>
> >Microscopy and Imaging Center
>
> >Texas A&M University
>
> >
>
> >
>
> >On Thu, 7 Jul 2011 13:20:02 -0600, 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
>
> >>*****
>
> >>
>
> >>If scattering is the issue then adaptive optics will be more advantageous
>
> >>than dispersion control.  The adaptive optics will help compensate
> somewhat
>
> >>for the scattering and aberrations induced by the tissue.  To get good 2P
>
> >>imaging you need a good focal spot more-so than you need a perfectly
>
> >>transform limited pulse.  Adaptive optics will help keep your focus
> together
>
> >>as you try to image deeply.  That said, dispersion compensation will help
>
> >>somewhat so if you already have the necessary equipment then try it.
>
> >>
>
> >>Craig
>
> >>
>
> >>
>
> >>
>
> >>On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
>
> >>[hidden email]> wrote:
>
> >>
>
> >>>  *****
>
> >>>  To join, leave or search the confocal microscopy listserv, go to:
>
> >>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> >>>  *****
>
> >>>
>
> >>>  Hi everybody,
>
> >>>  I am planning to image fluorescent neurons in vivo approximately  200
> um
>
> >>>  below the pia with a standard Ti:Sa laser.
>
> >>>  I wonder if there is a clear advantage to use pulse compression to
>
> >>>  compensate for dispersion of pulses due to tissue.
>
> >>>  I understand theoretical arguments in favor of pulse compression.
>
> >>>  However from an experimental point of view, are there some people here
>
> >in
>
> >>>  the list that have experienced some gain (in lowering the intensity of
> the
>
> >>>  exciting beam for example).
>
> >>>  Any comments would be greatly appreciated.
>
> >>>  Thanks a lot
>
> >>>  Stephane
>
> >>>
>
>
>
>
>
> --
>
> Jim Pawley (Summer address) c/o Postmaster,
>
> Egmont, BC, Canada, V0N-1N0 604-883-2095,
>
> [hidden email]
>
>
>
> -----
>
> No virus found in this message.
>
> Checked by AVG - www.avg.com
>
> Version: 10.0.1388 / Virus Database: 1516/3751 - Release Date: 07/08/11
>
>
>
>

[James Pawley]

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

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Is there a way to estimate how much of the
>damage comes from photochemistry and how much is
>heating? Is it possibe to measure temperature
>using fluorescence to determine if the sample is
>locally heated?
>
>best wishes
>
>Andreas
>

Dear Andreas,

In the early days of 2-photon, there were a lot
of calculations about heating and few suggested
even as much as one deg C (unless your specimen
has a high concentration of melanin or
chlorophyll or some major IR absorber, then
forget it).

So I think we can assume photochemistry but the
experiments you suggest are hard to do (The spot
gets distorted by RI inhomogeneities in the
specimen, so you don't know the intensity let
alone its square. And besides, there are
different intensities as you move away from
focus. And it depends on the amount of fluorophor
(damage proportional to emitted light) and a lot
of other factors.

There are two chapters on Phototoxicity in The
Handbook, but they are only a start....

Good luck,

JP

>
>
>-----Original Message-----
>From: Guy Cox <[hidden email]>
>To: CONFOCALMICROSCOPY <[hidden email]>
>Sent: Sat, 9 Jul 2011 9:16
>Subject: Re: Pulse compression and in vivo imaging
>
>
>*****
>
>To join, leave or search the confocal microscopy listserv, go to:
>
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>*****
>
>
>
>There are a lot of points here.  (Actually I suppose it should be 'There is a
>
>lot ....')
>
>
>
>Firstly, shorter pulses will not make the psf larger.  The psf depends on the
>
>wavelength, nothing else (and is, in principle, infinite ...)  The only thing
>
>that will make it appear to spread is if you are
>saturating the fluorescence at
>
>the centre of the psf, and this will apply
>irrespective of whether you are doing
>
>2P or regular confocal.  There are all sorts of
>reasons why saturation is a bad
>
>thing which we need not go into here.
>
>
>
>If you shorten the pulse while keeping the power
>the same you will increase all
>
>2 and 3 photon processes, both damaging and not.
>Since 2P follows a square law
>
>and 3P a cube law you will change the relative
>proportions, as Jim says. If you
>
>shorten the pulses and keep the peak intensity
>the same you will reduce overall
>
>power which at least will reduce heating of the sample so there should be some
>
>benefit, without affecting multi-photon processes.  I suspect that what most
>
>people do is something in between.
>
>
>
>Why is 2P more damaging?  Arguably it's not - Vadim Dedov and I were able to
>
>measure mitochondrial membrane potential in
>nerve cells with JC1 using 2-photon
>
>excitation while equivalent single-photon excitation killed the cells and we
>
>couldn't measure anything.  There are lots of
>other examples in the literature.
>
>What is true is that 2P can cause different sorts of damage.  The most extreme
>
>is breakdown caused by the electric field, which appears as bright flashes as
>
>you scan and 'craters' thereafter.  If you
>increase the peak electric field you
>
>will naturally increase this damage.
>
>
>
>Another point is more subtle.  Chemical selection rules state that in a
>
>symmetrical molecule, 2P excitation must occur to a different state than 1P.
>
>This means you will not excite the S1 state, and
>hence you have an enhanced rate
>
>of inter-system crossing into a triplet state.  This is a very noticeable with
>
>fluorescein, since it is symmetrical.  There are lots of published spectra out
>
>there now - if a fluorochrome shows very different 1P and 2P spectra you'd do
>
>best to avoid it.
>
>
>
>Finally, when we compress pulses we may not get what we think we are getting.
>
>Chirping gives a pulse a strange shape, which we
>hope will even out to a normal
>
>pulse after passing through our optics.  If in fact we excite with a chirped
>
>pulse then the peak intensity may be much high higher than we'd calculate from
>
>the nominal pulse length and average power.
>
>
>
> Guy
>
>
>
>
>
>Optical Imaging Techniques in Cell Biology
>
>by Guy Cox    CRC Press / Taylor & Francis
>
>      http://www.guycox.com/optical.htm
>
>______________________________________________
>
>Associate Professor Guy Cox, MA, DPhil(Oxon)
>
>Australian Centre for Microscopy & Microanalysis,
>
>Madsen Building F09, University of Sydney, NSW 2006
>
>
>
>Phone [phone]     Fax [phone]
>
>              Mobile [phone]
>
>______________________________________________
>
>       http://www.guycox.net
>
>
>
>
>
>-----Original Message-----
>
>From: Confocal Microscopy List [mailto:[hidden email]] On
>
>Behalf Of James Pawley
>
>Sent: Saturday, 9 July 2011 11:05 AM
>
>To: [hidden email]
>
>Subject: Re: Pulse compression and in vivo imaging
>
>
>
>*****
>
>To join, leave or search the confocal microscopy listserv, go to:
>
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>*****
>
>
>
>Hi all,
>
>
>
>Just a comment. Numerous studies on phototoxicity
>
>have shown that, in both single- and 2-photon
>
>microscopy, damage is (at least) proportional to
>
>the number of molecular excitations. If this
>
>holds, then if shorter pulses provide more
>
>(non-descanned) signal, it should also produce
>
>more photodamage.
>
>
>
>In addition, Dave Piston often made two points:
>
>that damage/excitation was often more severe with
>
>2-photon than single-photon excitation, and that,
>
>(depending on the wavelength) the shorter,
>
>higher-peak-power pulses that increase 2-photon
>
>signal may also increase 3-photon excitation of
>
>natural fluorophors in the cell.
>
>
>
>Have any of you noticed more photodamage when
>
>using shorter pulses? (Photodamage can cover a
>
>lot of effects from exploding cells to cells that
>
>should divide but fail to do so. Any change would
>
>be of interest to me.)
>
>
>
>Finally, more intense pulses means that the
>
>"threshold" for 2-photon excitation will be
>
>reached farther above and below the expected
>
>plane of focus than would be the case with
>
>longer, less intense pulses. i.e., at least some
>
>of the extra signal seen with shorter pulses may
>
>be the result of the PSF being larger in x,y and
>
>z, meaning that you excite more dye molecules.
>
>(As one moves above or below the focus plane, the
>
>hour-glass PSF becomes wider as well as taller.)
>
>
>
>Has anyone seen a change in resolution when using shorter pulses?
>
>
>
>Cheers,
>
>
>
>Jim Pawley
>
>
>
>
>
>>*****
>
>>To join, leave or search the confocal microscopy listserv, go to:
>
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>>*****
>
>>
>
>>Also, it depends on the pulse width.
>
>>the shorter the pulse, the more you may need the dispersion control as you go
>
>>deeper in the sample.
>
>>On our system with 10 fs pulses, we really cannot live without pre-chirp
>
>>(dispersion control). Your standard oscillator (~100-fs pulses?) is much more
>
>>forgiving.
>
>>
>
>>Stan Vitha
>
>>Microscopy and Imaging Center
>
>>Texas A&M University
>
>>
>
>>
>
>>On Thu, 7 Jul 2011 13:20:02 -0600, 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
>
>>>*****
>
>>>
>
>>>If scattering is the issue then adaptive optics will be more advantageous
>
>>>than dispersion control.  The adaptive optics will help compensate somewhat
>
>>>for the scattering and aberrations induced by the tissue.  To get good 2P
>
>>>imaging you need a good focal spot more-so than you need a perfectly
>
>>>transform limited pulse.  Adaptive optics will help keep your focus together
>
>>>as you try to image deeply.  That said, dispersion compensation will help
>
>>>somewhat so if you already have the necessary equipment then try it.
>
>>>
>
>>>Craig
>
>>>
>
>>>
>
>>>
>
>>>On Thu, Jul 7, 2011 at 4:44 AM, Stéphane Pagès <
>
>>>[hidden email]> wrote:
>
>>>
>
>>>>   *****
>
>>>>   To join, leave or search the confocal microscopy listserv, go to:
>
>>>>   http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>>>>   *****
>
>>>>
>
>>>>   Hi everybody,
>
>>>>   I am planning to image fluorescent neurons in vivo approximately  200 um
>
>>>>   below the pia with a standard Ti:Sa laser.
>
>>>>   I wonder if there is a clear advantage to use pulse compression to
>
>>>>   compensate for dispersion of pulses due to tissue.
>
>>>>   I understand theoretical arguments in favor of pulse compression.
>
>>>>   However from an experimental point of view, are there some people here
>
>>in
>
>>>>   the list that have experienced some gain
>>>>(in lowering the intensity of the
>
>>>>   exciting beam for example).
>
>>>>   Any comments would be greatly appreciated.
>
>>>>   Thanks a lot
>
>>>>   Stephane
>
>>>>
>
>
>
>
>
>--
>
>Jim Pawley (Summer address) c/o Postmaster,
>
>Egmont, BC, Canada, V0N-1N0 604-883-2095,
>
>[hidden email]
>
>
>
>-----
>
>No virus found in this message.
>
>Checked by AVG - www.avg.com
>
>Version: 10.0.1388 / Virus Database: 1516/3751 - Release Date: 07/08/11
>
>
>

--
Jim Pawley (Summer address) c/o Postmaster,
Egmont, BC, Canada, V0N-1N0 604-883-2095,
[hidden email]

[James Pawley]

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

Following on from the messages Jim and I posted
on the list on 'chirping' we had a lengthy
off-list exchange, and we thought that it might
be of interest to others, so here is the
discussion.  Jim is in italics, I'm in normal
type.

 
Guy


Firstly, shorter pulses will not make the psf
larger.  The psf depends on the wavelength,
nothing else (and is, in principle, infinite ...)
The only thing that will make it appear to spread
is if you are saturating the fluorescence at the
centre of the psf, and this will apply
irrespective of whether you are doing 2P or
regular confocal.  There are all sorts of reasons
why saturation is a bad thing which we need not
go into here.

I think you misunderstand me. I agree that the
"shape of the converging rays" does not depend on
the pulse length. However, these converging rays
represent a light intensity at each z plane.
Normally, we assume that this intensity only
reaches the instantaneous level needed to elicit
(much) fluorescence in the "blurry blob" near
crossover. Admittedly, this blob has fussy edges
but the does have a "z-resolution," in the form
of some Gaussian-ish response function.



                 This has nothing to do with
multiphoton or pulse length - there is no true
zero to the confocal psf in Z so very bright
objects will intrude into the planes where they
might not (naively) be expected.   I've seen this
effect any number of times in ordinary confocal
imaging.  You will find it mentioned in my book!
The only time the psf is actually affected,
though, is if we saturate in the centre - and
this applies whatever illumination mode we are
using.  I think we really agree about this.


Yes. And saturation at the peak is mentioned in my book.



However, it seems obvious to me that as the peak
power goes up, it will become high enough to
produce significant signal at some (short)
distance above or below the focus plane. I agree
that the in-focus plane will still produce more
more signal but as you mention, saturation is
possible (see duty cycle below) and I think that
the flattening caused in this way, coupled with
the effect just mentioned above, could reduce
z-res (depending on the actual peak intensity
levels).



Please recall, that my comment was addressed as a
possibility and asked is anyone had noticed this.


If you shorten the pulse while keeping the power
the same you will increase all 2 and 3 photon
processes, both damaging and not.  Since 2P
follows a square law and 3P a cube law you will
change the relative proportions, as Jim says. If
you shorten the pulses and keep the peak
intensity the same you will reduce overall power
which at least will reduce heating of the sample
so there should be some benefit, without
affecting multi-photon processes.  I suspect that
what most people do is something in between.

Surely, if you shorten pulses without increasing
peak power, you will reduce average excitation
(time x intensity squared) and have less signal.
I would guess that people seldom do this (except
to the extent that, when making the adjustments
for shorter pulses, you may affect the average
power inadvertently). They try to shorten pulses
while keeping AVERAGE power the same.



             OK, sloppy writing on my part.  I
should have said keeping average EXCITATION the
same.  But since excitation is intensity squared,
this is NOT keeping the average power the same.
 I think we are in total agreement about what
happens if they keep the average power the same.


Why is 2P more damaging?  Arguably it's not -
Vadim Dedov and I were able to measure
mitochondrial membrane potential in nerve cells
with JC1 using 2-photon excitation while
equivalent single-photon excitation killed the
cells and we couldn't measure anything.



But I didn't say that "2-p is more damaging". I
said very specifically that 2-photon was more
damaging per excitation. But as the excitations
are confined to near the focus plane, then, on
thickish specimens, there are far fewer total
excitations and therefore less damage overall.

  And mitochondria may be an anomalous test object
as they are set up to withstand singlet O2.



                 There are plenty of
non-mitochondrial examples.  Remember the famous
Cornell sea-urchin egg division series.  José
Feijó has shown this on many different botanical
examples.  How much damage is due to out of plane
excitation is hard to know,  though I suppose one
could use a very thin sample (film of bacteria,
for example) to test this.  But UV is so
obviously and immediately damaging to many living
cells that I find your thesis hard to sustain.



Once you get past 20µm in (assuming some stain
through out), I think that out of plane damage is
by far the predominant mechanism, especially on
the embryoes you mention. (Jayne Sparrow and the
mouse egg that hatched?)



  There are lots of other examples in the
literature.  What is true is that 2P can cause
different sorts of damage.  The most extreme is
breakdown caused by the electric field, which
appears as bright flashes as you scan and
'craters' thereafter.  If you increase the peak
electric field you will naturally increase this
damage.

  Another point is more subtle.  Chemical
selection rules state that in a symmetrical
molecule, 2P excitation must occur to a different
state than 1P.  This means you will not excite
the S1 state, and hence you have an enhanced rate
of inter-system crossing into a triplet state.
This is a very noticeable with fluorescein, since
it is symmetrical.  There are lots of published
spectra out there now - if a fluorochrome shows
very different 1P and 2P spectra you'd do best to
avoid it.

Finally, when we compress pulses we may not get
what we think we are getting.  Chirping gives a
pulse a strange shape, which we hope will even
out to a normal pulse after passing through our
optics.  If in fact we excite with a chirped
pulse then the peak intensity may be much high
higher than we'd calculate from the nominal pulse
length and average power.



One more factor. As 2p is pulsed, the duty cycle
is usually less than 10%. This means that people
often work nearer to singlet-state saturation
when using 2photon (to get an image in the same
scan time). This means that a lot more excited
molecules are present in the very high excitation
field near the centre of the focus, and increases
the likelihood of "one-plus-one" (or maybe 2 plus
one) overexcitation. Many smart, 2-photon folks
blame this for much of the increased
bleaching/excitation noted.



                 Well, they may be right but I'd
like to see evidence.  In all the cases I've
looked at where people reported rapid bleaching
in 2P it's been the selection rule issue that was
obviously to blame.  People think that because
FITC is such a super-bright and relatively stable
dye it must be the label of choice.  Then they
excite at about 750nm (where it does indeed have
a peak).  And it bleaches very fast indeed.  I
pointed this out at an FOM meeting more than 10
years ago, in discussion on a paper which had
measured FITC bleach rates in 2P.  Actually if
you excite FITC at 900nm it's much more stable.


I accept the point. And Piston did note extreme
differences in bleaching rates of different dyes.


                 The other curious feature I've
seen in postings on this issue is that most of
these people seem to be using 'experimental'
systems with nothing (other than adjusting the
laser) to control the power reaching the sample.
So their problems are hardly surprising!  Clearly
you need to use an EOM or whatever to control the
power.   You cannot adjust a Ti-S at all
satisfactorily by adjusting the input excitation
- I know, I've tried it.

Again, we agree.

--
Jim Pawley (Summer address) c/o Postmaster,
Egmont, BC, Canada, V0N-1N0 604-883-2095,
[hidden email]

[George McNamara]

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

Hi Wolfgang,

Check out:

Synchronously amplified fluorescence image recovery (SAFIRe).
</pubmed/19902923> Richards CI, Hsiang JC, Dickson RM. J Phys Chem B.
2010 Jan 14;114(1):660-5. PMID: 219902923 (dyes that form triplet state
readily at room temp.). Also cites some earlier work and they have a
later FRET-SAFIRe paper.

and

Arnold Giske's cryoSTED PhD dissertation

http://archiv.ub.uni-heidelberg.de/volltextserver/volltexte/2008/7969/pdf/CryoSTED_microscopy_PhDThesis_agiske.pdf
(if the pdf comes up blank initially, refresh the web page and/or scroll
down).

Dr. Giske mostly used ATTO 532 (which has low rate into triplet state(s)
at room temp), 671 nm for triplet depletion (Gaussian spot superimposed
on confocal excitation spot), and as the title indicates, cold.

I am wondering whether triplet state depletion is worth mentioning as a
potential opportunity to improve signal intensity for our STED proposal
(if we have the right laser). My current limited understanding is that
very good fluorophores will not benefit much compared to yet another
variable to confuse reviewers and (if we get the money) users.

Enjoy,

George


-------- Original Message --------
Subject: Re: Fwd: Re: Pulse compression and in vivo imaging
Date: Fri, 15 Jul 2011 09:31:47 +0200
From: Wolfgang Staroske <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
To: [hidden email]



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*****

Hi George,

that's a good point, I haven't thought about this possibility yet.

Destruction would mean excite the molecule into higher triplet states
until its is ionized.
Triplet state depletion would be something like stimulated emission
without emission of a photon.

I would favor the first one, because this process is quantum
mechanically allowed and the energies of the IR photons should be enough
to go up the electronic states.
For the triplet-state depletion, I think the probability is as low as
for the inter-system-crossing from the singlet to the triplet state and
additionally the energy of  the triplet state is probably higher than
the energy of the IR photon. Of course that could be a
two-photon-process with enough energy than in, which would probably
happen not in the same pulse but in the next ones. On the other hand I
never heard of triplet-state depletion in one-photon-excitation and if
you get the same signal in one and two-photon excitation the
probabilities of absorption / stimulated emission (of one or two photons
respectively) should be the same.

But at all I'm not an expert in quantum mechanics, the only fact I can
state is that in the case of two-photon excitation the triplet lifetime
is either very short (below time resolution<250ns) due to triplet state
depletion or infinite due to photobleaching.

Bye Wolfgang


Am 20:59, schrieb George McNamara:

>  *****
>  <br>To join, leave or search the confocal microscopy listserv, go to:
>  <br>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>  <br>*****
>  <br>
>  <br>Hi Wolfgang,
>  <br>
>  <br>why do you think you are causing photodestruction, as opposed to
>  triplet
>  state depletion back to the ground state?
>  <br>
>  <br>thanks,
>  <br>
>  <br>George
>  <br>
>  <br>
>  <br>On 7/14/2011 10:30 AM, Wolfgang Staroske wrote:
>  <br><blockquote type=cite>*****
>  <br>To join, leave or search the confocal microscopy listserv, go to:
>  <br>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>  <br>*****
>  <br>
>  <br>Dear all,
>  <br>
>  <br>Am 20:59, schrieb James Pawley:
>  <br><blockquote type=cite>&lt;br&gt;JP: One more factor. As 2p is
>  pulsed, the duty cycle
>  <br>&lt;br&gt;is usually less than 10%. This means that people
>  <br>&lt;br&gt;often work nearer to singlet-state saturation
>  <br>&lt;br&gt;when using 2photon (to get an image in the same
>  <br>&lt;br&gt;scan time). This means that a lot more excited
>  <br>&lt;br&gt;molecules are present in the very high excitation
>  <br>&lt;br&gt;field near the centre of the focus, and increases
>  <br>&lt;br&gt;the likelihood of "one-plus-one" (or maybe 2 plus
>  <br>&lt;br&gt;one?) overexcitation. Many smart, 2-photon folks
>  <br>&lt;br&gt;blame this for much of the increased
>  <br>&lt;br&gt;bleaching/excitation noted.
>  <br></blockquote>
>  <br>I would like to comment on this. In FCS Experiments we see that all
>  dyes, even the ones which show a strong triplet fluctuation in
>  one-photon excitation, show no triplet fluctuation in the case of
>  two-photon excitation.
>  <br>
>  <br>Our hypothesis for that is the following. The lifetime of the triplet
>  state is long enough, that each molecule, which entered the triplet
>  state, absorbs a third IR photon, which destroys the dye molecule. So
>  molecules which entered the triplet state are dark from this time
>  point on.
>  <br>
>  <br>In imaging of course this possibility is reduced because the laser is
>  scanned and pixel dwell times are usually in the range or below the
>  triplet state lifetime (few µs), while in FCS the residence time of
>  even small molecules are at least 20µs.
>  <br>
>  <br>Bye Wolfgang
>  <br>
>  <br>
>  <br></blockquote>
>  <br>
>  <br></body>
>  </html>
>  </html>

--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

Technische Universität Dresden
Biotechnology Center
Tatzberg 47/49
01307 Dresden, Germany

Tel.: [phone]
Fax.: [phone]
E-Mail: [hidden email]
Webpage: www.biotec.tu-dresden.de

[Wolfgang Staroske]

*****
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*****

Hi George,

thanks for your literature. I checked both, but concentrated more on
Arnolds PhD thesis. (I know him personally, as he works now as CW-STED
developer with Leica, and we had such a demo here, with a rather
problematic start)

In the paper they don't say anything about the electronic states behind.
Arnold suggests that the depopulation goes T1 -> Tn -> Sn -> S1 -> S0.
The same mechanism which I suggested for the bleaching (T1 -> Tn ->
dead). In the end I think it is intensity dependent, because the rate
for ISC from Tn to Sn competes against photon driven ionization from Tn.
There is a nice graph in his thesis, where it is visible that there is
an optimal T-depopulation laser power, and with higher laser powers the
fluorescence decreases again, I think due to photobleaching. But in
2-Photon-excitation you have no control on that laser power, because it
is your pulsed IR laser (Arnold had 300mW cw @ 671nm, our 2-Photon-laser
has 300mW averaged in several hundred fs @ 80MHz - I think a rather
common system).  Additionally he clearly states, that this method
doesn't work at room temperature and T-REX (Donnert et al., PNAS 103,
p10440) has to be used, which is in principle only waiting. So I still
would bet for bleaching at room temperature.

Of course the only way to figure that out, is to measure it in a pulsed
regime as in Arnolds thesis. First excite the dye of interest with a
pulsed one-photon laser and record the fluorescence. Then do the same
but use your two-photon pulse after some ns (fluorescene should be
decayed) as a second pulse for T-depletion or bleaching, and compare
both traces. Of course in second run, record only the fluorescence  from
the one-photon excitation. T-Depletion should increase the fluorescence
signal, while photobleaching should decrease it.

Bye Wolfgang


  Am 20:59, schrieb George McNamara:

> *****
> <br>To join, leave or search the confocal microscopy listserv, go to:
> <br>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> <br>*****
> <br>
> <br>Hi Wolfgang,
> <br>
> <br>Check out:
> <br>
> <br>Synchronously amplified fluorescence image recovery (SAFIRe).
> &lt;/pubmed/19902923&gt; Richards CI, Hsiang JC, Dickson RM. J Phys
> Chem B.
> 2010 Jan 14;114(1):660-5. PMID: 219902923 (dyes that form triplet state
> readily at room temp.). Also cites some earlier work and they have a
> later FRET-SAFIRe paper.
> <br>
> <br>and
> <br>
> <br>Arnold Giske's cryoSTED PhD dissertation
> <br>
> <br>http://archiv.ub.uni-heidelberg.de/volltextserver/volltexte/2008/7969/pdf/CryoSTED_microscopy_PhDThesis_agiske.pdf
> <br>(if the pdf comes up blank initially, refresh the web page and/or
> scroll
> down).
> <br>
> <br>Dr. Giske mostly used ATTO 532 (which has low rate into triplet
> state(s)
> at room temp), 671 nm for triplet depletion (Gaussian spot superimposed
> on confocal excitation spot), and as the title indicates, cold.
> <br>
> <br>I am wondering whether triplet state depletion is worth mentioning
> as a
> potential opportunity to improve signal intensity for our STED proposal
> (if we have the right laser). My current limited understanding is that
> very good fluorophores will not benefit much compared to yet another
> variable to confuse reviewers and (if we get the money) users.
> <br>
> <br>Enjoy,
> <br>
> <br>George
> <br>
> <br>
> <br>-------- Original Message --------
> <br>Subject:&nbsp;&nbsp;&nbsp;&nbsp; Re: Fwd: Re: Pulse compression
> and in vivo imaging
> <br>Date:&nbsp;&nbsp;&nbsp;&nbsp; Fri, 15 Jul 2011 09:31:47 +0200
> <br>From:&nbsp;&nbsp;&nbsp;&nbsp; Wolfgang Staroske
> &lt;[hidden email]&gt;
> <br>Reply-To:&nbsp;&nbsp;&nbsp;&nbsp; Confocal Microscopy List
> &lt;[hidden email]&gt;
> <br>To:&nbsp;&nbsp;&nbsp;&nbsp; [hidden email]
> <br>
> <br>
> <br>
> <br>*****
> <br>To join, leave or search the confocal microscopy listserv, go to:
> <br>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> <br>*****
> <br>
> <br>Hi George,
> <br>
> <br>that's a good point, I haven't thought about this possibility yet.
> <br>
> <br>Destruction would mean excite the molecule into higher triplet states
> <br>until its is ionized.
> <br>Triplet state depletion would be something like stimulated emission
> <br>without emission of a photon.
> <br>
> <br>I would favor the first one, because this process is quantum
> <br>mechanically allowed and the energies of the IR photons should be
> enough
> <br>to go up the electronic states.
> <br>For the triplet-state depletion, I think the probability is as low as
> <br>for the inter-system-crossing from the singlet to the triplet
> state and
> <br>additionally the energy of&nbsp; the triplet state is probably
> higher than
> <br>the energy of the IR photon. Of course that could be a
> <br>two-photon-process with enough energy than in, which would probably
> <br>happen not in the same pulse but in the next ones. On the other hand I
> <br>never heard of triplet-state depletion in one-photon-excitation and if
> <br>you get the same signal in one and two-photon excitation the
> <br>probabilities of absorption / stimulated emission (of one or two
> photons
> <br>respectively) should be the same.
> <br>
> <br>But at all I'm not an expert in quantum mechanics, the only fact I can
> <br>state is that in the case of two-photon excitation the triplet
> lifetime
> <br>is either very short (below time resolution&lt;250ns) due to
> triplet state
> <br>depletion or infinite due to photobleaching.
> <br>
> <br>Bye Wolfgang
> <br>
> <br>
> <br>Am 20:59, schrieb George McNamara:
> <br><blockquote type=cite>&nbsp;*****
> <br>&nbsp;&lt;br&gt;To join, leave or search the confocal microscopy
> listserv, go to:
> <br>&nbsp;&lt;br&gt;http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> <br>&nbsp;&lt;br&gt;*****
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;Hi Wolfgang,
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;why do you think you are causing photodestruction,
> as opposed to
> <br>&nbsp;triplet
> <br>&nbsp;state depletion back to the ground state?
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;thanks,
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;George
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;On 7/14/2011 10:30 AM, Wolfgang Staroske wrote:
> <br>&nbsp;&lt;br&gt;&lt;blockquote type=cite&gt;*****
> <br>&nbsp;&lt;br&gt;To join, leave or search the confocal microscopy
> listserv, go to:
> <br>&nbsp;&lt;br&gt;http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> <br>&nbsp;&lt;br&gt;*****
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;Dear all,
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;Am 20:59, schrieb James Pawley:
> <br>&nbsp;&lt;br&gt;&lt;blockquote type=cite&gt;&amp;lt;br&amp;gt;JP:
> One more factor. As 2p is
> <br>&nbsp;pulsed, the duty cycle
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;is usually less than 10%. This
> means that people
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;often work nearer to
> singlet-state saturation
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;when using 2photon (to get an
> image in the same
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;scan time). This means that a
> lot more excited
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;molecules are present in the
> very high excitation
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;field near the centre of the
> focus, and increases
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;the likelihood of "one-plus-one"
> (or maybe 2 plus
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;one?) overexcitation. Many
> smart, 2-photon folks
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;blame this for much of the increased
> <br>&nbsp;&lt;br&gt;&amp;lt;br&amp;gt;bleaching/excitation noted.
> <br>&nbsp;&lt;br&gt;&lt;/blockquote&gt;
> <br>&nbsp;&lt;br&gt;I would like to comment on this. In FCS
> Experiments we see that all
> <br>&nbsp;dyes, even the ones which show a strong triplet fluctuation in
> <br>&nbsp;one-photon excitation, show no triplet fluctuation in the
> case of
> <br>&nbsp;two-photon excitation.
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;Our hypothesis for that is the following. The
> lifetime of the triplet
> <br>&nbsp;state is long enough, that each molecule, which entered the
> triplet
> <br>&nbsp;state, absorbs a third IR photon, which destroys the dye
> molecule. So
> <br>&nbsp;molecules which entered the triplet state are dark from this
> time
> <br>&nbsp;point on.
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;In imaging of course this possibility is reduced
> because the laser is
> <br>&nbsp;scanned and pixel dwell times are usually in the range or
> below the
> <br>&nbsp;triplet state lifetime (few µs), while in FCS the residence
> time of
> <br>&nbsp;even small molecules are at least 20µs.
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;Bye Wolfgang
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;&lt;/blockquote&gt;
> <br>&nbsp;&lt;br&gt;
> <br>&nbsp;&lt;br&gt;&lt;/body&gt;
> <br>&nbsp;&lt;/html&gt;
> <br>&nbsp;&lt;/html&gt;
> <br></blockquote>
> <br></body>
> </html>
> </html>

--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

Technische Universität Dresden
Biotechnology Center
Tatzberg 47/49
01307 Dresden, Germany

Tel.: [phone]
Fax.: [phone]
E-Mail: [hidden email]
Webpage: www.biotec.tu-dresden.de