http://confocal-microscopy-list.275.s1.nabble.com/Pulse-compression-and-in-vivo-imaging-tp6557894p6567011.html
alone its square. And besides, there are
focus. And it depends on the amount of fluorophor
>
>
>-----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
>
>
>*****
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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:
[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]
>
>
>
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