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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 <[log in to unmask]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
> 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 <[log in to unmask]>
> To: CONFOCALMICROSCOPY <[log in to unmask]>
> Sent: Sat, 9 Jul 2011 9:16
> Subject: Re: Pulse compression and in vivo imaging
>
>
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> To join, leave or search the confocal microscopy listserv, go to:
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
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>
>
> 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 +61 2 9351 3176     Fax +61 2 9351 7682
>
>             Mobile 0413 281 861
>
> ______________________________________________
>
>      http://www.guycox.net
>
>
>
>
>
> -----Original Message-----
>
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On
>
> Behalf Of James Pawley
>
> Sent: Saturday, 9 July 2011 11:05 AM
>
> To: [log in to unmask]
>
> 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
>
> ><[log in to unmask]> wrote:
>
> >
>
> >>*****
>
> >>To join, leave or search the confocal microscopy listserv, go to:
>
> >>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> >>*****
>
> >>
>
> >>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 <
>
> >>[log in to unmask]> wrote:
>
> >>
>
> >>>  *****
>
> >>>  To join, leave or search the confocal microscopy listserv, go to:
>
> >>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> >>>  *****
>
> >>>
>
> >>>  Hi 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,
>
> [log in to unmask]
>
>
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