http://confocal-microscopy-list.275.s1.nabble.com/Pulse-compression-and-in-vivo-imaging-tp6557894p6565095.html
Tsunami nearly transform limited at the sample. They are about 60fs when
transform limited. The first thing we noticed was a significant increase in
power turned down to minimum. We did get an order of magnitude increase is
signal, but the photodamage effects made things impractical. After some
at reasonable powers with minimum tissue destruction. Our samples were
the environment for free radical generation. Your mileage may vary by
sample. As for resolution, no particular improvement was noticed.
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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].**ca <
[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,
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>