Suitable laser for 2-photon brain imaging

[Hana Uhlirova]

*****
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

[Craig Brideau]

*****
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 Smith]

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

[Jakub Chojnacki]

*****
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,

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

--
Dr Jakub Chojnacki
IrsiCaixa AIDS Research Institute
Barcelona, Spain
[hidden email] <https://twitter.com/IrsiCaixa>

[0000001ed7f52e4a-dmarc-request]

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

[Thomas Abraham]

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

[Mark Cannell-2]

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

[Craig Brideau]

*****
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.
*****

Many of the comments here have revolved around pulse dispersion, but in my
own experience this is not the main factor which limits how deep you can
image using 2P. Unless your laser has an absurdly wide bandwidth you will
not experience much dispersion unless you are going through many tens of mm
of material. The largest contributor to dispersion will be the microscope
itself: The 1-3mm of tissue you penetrate will have a minuscule impact in
comparison.
The largest contribution to depth in 2P is scattering and RI mismatch. If
the tissue is very heterogeneous the light is scattered and the tight focus
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
option to avoid this is to use higher-energy pulses (not the same as higher
average power so be careful of this!) or some form of wavefront correction
(complex!) to compensate for the scatter. More simply, working with longer
wavelengths reduces scatter in a passive way and is less complicated to
implement. Do be aware that there are water absorption peaks around 1200nm
that you will want to avoid, and it may take some work to find suitable
NIR-absorbing fluorophores compatible with the wavelength you decide to
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
far red.

I hope you find this helpful.
Craig

On Sun, May 13, 2018 at 1:45 AM Mark Cannell <[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.
> *****
>
> 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  [phone]
>     > Fax [phone]
>     > e-mail: [hidden email]
>     > http://vision.berkeley.edu/?page_id=5635 <
> http://vision.berkeley.edu/>
>
>
>

[Hana Uhlirova]

*****
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.
*****

Many thanks to everyone for the explanations and the links that provide good and clear information about the topic.

Best regards, Hana

Hana Uhlirova
Institute of Scientific Instruments of the CAS
Czech Republic