http://confocal-microscopy-list.275.s1.nabble.com/Suitable-laser-for-2-photon-brain-imaging-tp7588232p7588250.html
image using 2P. Unless your laser has an absurdly wide bandwidth you will
of material. The largest contributor to dispersion will be the microscope
The largest contribution to depth in 2P is scattering and RI mismatch. If
required for 2P is broken up. This also limits the amount of laser energy
which reaches the focus, leading to overall reduced signal. Your best
(complex!) to compensate for the scatter. More simply, working with longer
implement. Do be aware that there are water absorption peaks around 1200nm
use. You will also need NIR detection, so red-extended PMTs will be
important. Note that GaAsP detectors typically do not perform well in the
> *****
> 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.
> *****
>
> You can correct most of the pulse dispersion empirically, just change the
> pre-chirp until the signal is maximised: Soeller C, Cannell MB.
> Construction of a two-photon microscope and optimisation of illumination
> pulse duration. Pflugers Arch. 1996;432:555–561.
>
> Mark B. Cannell. Ph.D. FRSNZ FISHR
> Department of Physiology, Pharmacology & Neuroscience
> School of Medical Sciences
> University Walk
> Bristol BS8 1TD
>
>
[hidden email]
>
>
>
> On 12/05/18, 9:31 PM, "Confocal Microscopy List on behalf of Thomas
> Abraham" <
[hidden email] on behalf of
[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.
> *****
>
> In multiphooton microscopy, there is average power and the peak
> power, and the latter depends on the pulse width in time domain and the
> pulse repetition rate. I have used Spectra Physics Tsunami (no GVD
> correction) and the DeepSee Insight (with GVD correction). I hardly notice
> any difference. Of course, the new system corrects the GVD in optical
> elements like in objective lens, but not within tissues which is far more
> complex to understand and estimate! Also, understand that it is not just
> excitation, the emission which is still in visible region is subjected to
> scattering and the depth of the imaging.
> > On May 12, 2018, at 12:24 PM, Andreas Bruckbauer <
>
[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.
> > *****
> >
> >
> > A large contribution to pulse broadening is caused by components
> like AOMs for intensity control, so it depends on the microscope used. You
> only need the full power if you are imaging at the far end of the spectrum,
> at 800 nm you would typically only use a few percent of the power for live
> imaging.
> >
> > best wishes
> >
> > Andreas
> >
> >
> > -----Original Message-----
> > From: Benjamin E Smith <
[hidden email]>
> > To: CONFOCALMICROSCOPY <
[hidden email]>
> > Sent: Fri, 11 May 2018 18:15
> > Subject: Re: Suitable laser for 2-photon brain imaging
> >
> > *****
> > 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 <
>
[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.
> >> *****
> >>
> >> 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 (510) 642-9712
> > Fax (510) 643-6791
> > e-mail:
[hidden email]
> >
http://vision.berkeley.edu/?page_id=5635 <
>
http://vision.berkeley.edu/>
>
>
>