http://confocal-microscopy-list.275.s1.nabble.com/Suitable-laser-for-2-photon-brain-imaging-tp7588232p7588247.html
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:
>
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> *****
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>
> 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
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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:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> Post images on
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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 (510) 642-9712
> Fax (510) 643-6791
> e-mail:
[hidden email]
>
http://vision.berkeley.edu/?page_id=5635 <
http://vision.berkeley.edu/>