Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Pulse-compression-and-in-vivo-imaging-tp6557894p6564610.html
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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:
>
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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:
>>
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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
>>>
--
Jim Pawley (Summer address) c/o Postmaster,
Egmont, BC, Canada, V0N-1N0 604-883-2095,
[hidden email]