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Hello,

Since I had a similar questions in mind, could I add one more question to
this topic of in vivo 2P imaging. I am also currently considering Chameleon
Discovery for live in vivo two color 2P experiments with standard
fluoroscopes or fluorescence proteins.
Would this 2P source be sufficient for this application or would a
different model be recommended?

Thank you,
Jakub



On 11 May 2018 at 19:13, Benjamin E Smith <[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
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> A prism compressor should offset the loss of power due to dispersion
> through the optics.  For example, if you send a 140 fs pulse through a
> standard scan lens, tube lens and objective, the pulse will spread out to
> about 500 fs due to group velocity dispersion (although this will vary
> significantly depending on the objective, tube lens, and scan lens - see
> the following paper: https://goo.gl/SsQYvQ).  The contribution of water
> and
> tissue is practically non-existent (for example 2mm of water will cause a
> 140 fs pulse to spread out to 140.0035 fs according to this paper:
> https://goo.gl/5NhHxF).
>
> Since the 140 fs pulse has spread out to a 500 fs pulse, the effective
> power density relative to the original pulse is 140/500 = 28%.  Therefore,
> in a very simplified scenario, if you start with a 4W 140 fs pulse, after
> all the optics, you now have the equivalent power density of a 1W 140 fs
> pulse (4W * 0.28), but still with all the heating of a 4W beam.  By using a
> prism compressor, assuming it is optimally tuned, you will get a 140 fs
> pulse at the sample.  With this simplified scenario, a 1W 140 fs laser with
> a prism compressor is equivalent to a 4W 140 fs laser without a prism
> compressor, but with 1/4 the heating so all in all the laser with the
> compressor is theoretically the superior setup (as long as the compressor
> is used correctly).  There are many papers that show reality diverges
> somewhat from theory, but that is to be expected with the optical
> complexity of biological samples paired with the non-linearity of 2P
> excitation.
>
> As far as FLIM goes, either laser will work equally well.  Even a 500 fs
> pulse is effectively instantaneous for a FLIM detector (which usually have
> temporal resolutions down to about 100 ps in ideal conditions), so both
> will look identical to a FLIM system.
>
> Hope this helps,
>    Ben Smith
>
>
>
>
>
> On Fri, May 11, 2018 at 9:24 AM, 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
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Hana, this depends heavily on what fluorophore you are using, whether
> > the sample is 'live' or not, etc. What is your situation?
> >
> > Craig
> >
> > On Fri, May 11, 2018 at 5:24 AM Hana Uhlirova <[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
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Hello list, I'd like to know your opinions about the laser type
> suitable
> > > for 2-photon in vivo brain imaging. We are considering the Chameleon
> > Ultra
> > > family from Coherent (Ultra, Ultra I and Ultra II) and Chameleon
> Vision.
> > > With Vision we would get the dispersion pre-compensation but the peak
> > power
> > > is only 2.5 W as is for the Ultra. Ultra I has peak power of 2.9 W and
> > > Ultra II 3.5 W. In my old lab we used to have the Ultra II which I
> think
> > is
> > > the most common choice.
> > > My questions:
> > > 1. Does anyone use Ultra or Ultra I for multi-photon in vivo brain
> > imaging
> > > and is the laser power sufficient?
> > > 2. Does someone use the dispersion pre-compensation of Vision I? If so,
> > in
> > > which microscope and does it provide a significant improvement over a
> > > non-compensated beam?
> > > 3. What are the implications of the laser choice on fluorescence
> lifetime
> > > imaging?
> > >
> > > Thank you very much for your opinions and suggestions.
> > >
> > > Hana Uhlirova
> > > Institute of Scientific Instruments of the CAS
> > > Czech Republic
> > >
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  (510) 642-9712
> Fax (510) 643-6791
> e-mail: [log in to unmask]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>



-- 
Dr Jakub Chojnacki
IrsiCaixa AIDS Research Institute
Barcelona, Spain
[log in to unmask] <https://twitter.com/IrsiCaixa>