Posted by
Andreas Bruckbauer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Pulse-compression-and-in-vivo-imaging-tp6557894p6566389.html
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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 <
[hidden email]>
To: CONFOCALMICROSCOPY <
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Sent: Sat, 9 Jul 2011 9:16
Subject: Re: Pulse compression and in vivo imaging
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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
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-----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
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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
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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:
>>
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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
>>>
--
Jim Pawley (Summer address) c/o Postmaster,
Egmont, BC, Canada, V0N-1N0 604-883-2095,
[hidden email]
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