Re: Suitable laser for 2-photon brain imaging

Posted by Benjamin Smith on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Suitable-laser-for-2-photon-brain-imaging-tp7588232p7588242.html

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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 <[hidden email]>
wrote:

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>
> 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 <[hidden email]>
> 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  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>