2P for zebrafish axon ablation

[Greg Ning]

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Dear all,

We are in process of acquiring a 2P microscope. One of the applications is to cut zebrafish axon while imaging. We failed to cut nerves with a demo system equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300 nm plus a fixed line at 1045 nm). The attempt was done with a 25x water dipping lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep. I have to say the demo test was done in a relative short time on a new system which we don't know much. My questions are -

  *   Any of you there have done 2p ablation with young/adult fish nerve? If so what equipment, laser, and parameters did you use to cut?
  *   Any of you there have done 2p ablation with a water dipping lens without coverslip?
  *   Any opinion about the laser for ablation, Spectra Physics vs Coherent?

Thank you very much in advance, and stay safe and healthy!

Greg


Gang (Greg) Ning

Microscopy Facility

Huck Institutes of the Life Sciences

Penn State University

MSC N-048

University Park, PA 16802

814-863-0994

https://www.huck.psu.edu/core-facilities/microscopy-facility

[Marc Reinig-2]

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You don’t mention what wavelength you were using. Have you tried using
deuterium for dipping? It’s less absorptive than water.


On Wed, Apr 29, 2020 at 07:10 Ning, Gang <[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.
> *****
>
> Dear all,
>
> We are in process of acquiring a 2P microscope. One of the applications is
> to cut zebrafish axon while imaging. We failed to cut nerves with a demo
> system equipped with Spectra Physics Insight X3 DUAL (tunable from 680 –
> 1300 nm plus a fixed line at 1045 nm). The attempt was done with a 25x
> water dipping lens NA1.0 without coverslip on a 7-day-old fish at about 750
> um deep. I have to say the demo test was done in a relative short time on a
> new system which we don't know much. My questions are -
>
>   *   Any of you there have done 2p ablation with young/adult fish nerve?
> If so what equipment, laser, and parameters did you use to cut?
>   *   Any of you there have done 2p ablation with a water dipping lens
> without coverslip?
>   *   Any opinion about the laser for ablation, Spectra Physics vs
> Coherent?
>
> Thank you very much in advance, and stay safe and healthy!
>
> Greg
>
>
> Gang (Greg) Ning
>
> Microscopy Facility
>
> Huck Institutes of the Life Sciences
>
> Penn State University
>
> MSC N-048
>
> University Park, PA 16802
>
> 814-863-0994
>
> https://www.huck.psu.edu/core-facilities/microscopy-facility
>

--
Marco

Sent from my iPhone

[Greg Ning]

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The laser wavelength was 780 nm.


Gang (Greg) Ning

Microscopy Facility

Huck Institutes of the Life Sciences

Penn State University

MSC N-048

University Park, PA 16802

814-863-0994

https://www.huck.psu.edu/core-facilities/microscopy-facility



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Marc Reinig <[hidden email]>
Sent: Wednesday, April 29, 2020 10:48 AM
To: [hidden email] <[hidden email]>
Subject: Re: 2P for zebrafish axon ablation

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You don’t mention what wavelength you were using. Have you tried using
deuterium for dipping? It’s less absorptive than water.


On Wed, Apr 29, 2020 at 07:10 Ning, Gang <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7Cgxn7%40PSU.EDU%7C42e3530451734bdc24a108d7ec4c92d4%7C7cf48d453ddb4389a9c1c115526eb52e%7C0%7C0%7C637237685933002136&amp;sdata=BS78GBZX5v9VkoLwgLscOzQFK4lJ3K9rPOo%2FqHCXj2Y%3D&amp;reserved=0
> Post images on https://nam01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=02%7C01%7Cgxn7%40PSU.EDU%7C42e3530451734bdc24a108d7ec4c92d4%7C7cf48d453ddb4389a9c1c115526eb52e%7C0%7C0%7C637237685933002136&amp;sdata=iOcNVL3IQ%2FkY61ucvrLjryXHC8PisQNKWULFt0u960M%3D&amp;reserved=0 and include the link in your posting.
> *****
>
> Dear all,
>
> We are in process of acquiring a 2P microscope. One of the applications is
> to cut zebrafish axon while imaging. We failed to cut nerves with a demo
> system equipped with Spectra Physics Insight X3 DUAL (tunable from 680 –
> 1300 nm plus a fixed line at 1045 nm). The attempt was done with a 25x
> water dipping lens NA1.0 without coverslip on a 7-day-old fish at about 750
> um deep. I have to say the demo test was done in a relative short time on a
> new system which we don't know much. My questions are -
>
>   *   Any of you there have done 2p ablation with young/adult fish nerve?
> If so what equipment, laser, and parameters did you use to cut?
>   *   Any of you there have done 2p ablation with a water dipping lens
> without coverslip?
>   *   Any opinion about the laser for ablation, Spectra Physics vs
> Coherent?
>
> Thank you very much in advance, and stay safe and healthy!
>
> Greg
>
>
> Gang (Greg) Ning
>
> Microscopy Facility
>
> Huck Institutes of the Life Sciences
>
> Penn State University
>
> MSC N-048
>
> University Park, PA 16802
>
> 814-863-0994
>
> https://www.huck.psu.edu/core-facilities/microscopy-facility
>

--
Marco

Sent from my iPhone

[Douglas Richardson]

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Hello Greg,

We've ablated cells in zebrafish with an Insight DS (the predecessor to the
X3 with less power), a 20x 1.0 water dipping objective, wavelengths 800-900
nm. It wasn't easy though.

I know of other groups who have compared the X3 to the MaiTai HP and found
the shorter pulse width (80fs vs. up to 200fs) improves ablation ability.

Hope that helps.

-Doug



On Wed, Apr 29, 2020 at 10:10 AM Ning, Gang <[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.
> *****
>
> Dear all,
>
> We are in process of acquiring a 2P microscope. One of the applications is
> to cut zebrafish axon while imaging. We failed to cut nerves with a demo
> system equipped with Spectra Physics Insight X3 DUAL (tunable from 680 –
> 1300 nm plus a fixed line at 1045 nm). The attempt was done with a 25x
> water dipping lens NA1.0 without coverslip on a 7-day-old fish at about 750
> um deep. I have to say the demo test was done in a relative short time on a
> new system which we don't know much. My questions are -
>
>   *   Any of you there have done 2p ablation with young/adult fish nerve?
> If so what equipment, laser, and parameters did you use to cut?
>   *   Any of you there have done 2p ablation with a water dipping lens
> without coverslip?
>   *   Any opinion about the laser for ablation, Spectra Physics vs
> Coherent?
>
> Thank you very much in advance, and stay safe and healthy!
>
> Greg
>
>
> Gang (Greg) Ning
>
> Microscopy Facility
>
> Huck Institutes of the Life Sciences
>
> Penn State University
>
> MSC N-048
>
> University Park, PA 16802
>
> 814-863-0994
>
> https://www.huck.psu.edu/core-facilities/microscopy-facility
>

[Craig Brideau]

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*****

About 10 years ago I ablated focal lesions in spinal cord with an old
Spectra Physics Tsunami, which is pretty similar to contemporary Ti:Saph
lasers. I believe the wavelength I used was 900nm, and I
dispersion-optimized the system for approximately 200fs pulse width and
used 10's of mW on the sample. My dwell time was fairly long, on the order
of 50-250 microseconds I believe. As Doug mentioned, shorter pulse widths
will improve efficacy, so consider a pulse compressor.

Craig

On Wed, Apr 29, 2020 at 9:18 AM Douglas Richardson <[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 Greg,
>
> We've ablated cells in zebrafish with an Insight DS (the predecessor to the
> X3 with less power), a 20x 1.0 water dipping objective, wavelengths 800-900
> nm. It wasn't easy though.
>
> I know of other groups who have compared the X3 to the MaiTai HP and found
> the shorter pulse width (80fs vs. up to 200fs) improves ablation ability.
>
> Hope that helps.
>
> -Doug
>
>
>
> On Wed, Apr 29, 2020 at 10:10 AM Ning, Gang <[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.
> > *****
> >
> > Dear all,
> >
> > We are in process of acquiring a 2P microscope. One of the applications
> is
> > to cut zebrafish axon while imaging. We failed to cut nerves with a demo
> > system equipped with Spectra Physics Insight X3 DUAL (tunable from 680 –
> > 1300 nm plus a fixed line at 1045 nm). The attempt was done with a 25x
> > water dipping lens NA1.0 without coverslip on a 7-day-old fish at about
> 750
> > um deep. I have to say the demo test was done in a relative short time
> on a
> > new system which we don't know much. My questions are -
> >
> >   *   Any of you there have done 2p ablation with young/adult fish nerve?
> > If so what equipment, laser, and parameters did you use to cut?
> >   *   Any of you there have done 2p ablation with a water dipping lens
> > without coverslip?
> >   *   Any opinion about the laser for ablation, Spectra Physics vs
> > Coherent?
> >
> > Thank you very much in advance, and stay safe and healthy!
> >
> > Greg
> >
> >
> > Gang (Greg) Ning
> >
> > Microscopy Facility
> >
> > Huck Institutes of the Life Sciences
> >
> > Penn State University
> >
> > MSC N-048
> >
> > University Park, PA 16802
> >
> > 814-863-0994
> >
> > https://www.huck.psu.edu/core-facilities/microscopy-facility
> >
>

[Kalpana Iyengar]

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**Commercial Response**

Hello Greg,

I want to direct you to a couple of publications in which the researchers use the Andor Micropoint laser system for axon ablation at 440 nm:
doi:10.1242/dev.004267
doi:10.1038/nn1803

We have an excellent photo-ablation system that has been very successful in similar experiments and can be fully automated. It can also be used in conjunction with any microscope system.

If you would like to learn more, I invite you to schedule a time to chat with us here: https://doodle.com/poll/chqeceehnnu67un6 <https://doodle.com/poll/chqeceehnnu67un6>

Hope this informs. Stay safe!

Best,
Kalpana Iyengar, Ph.D.
Confocal Applications Scientist
Andor Technology
[phone]
[hidden email] <mailto:[hidden email]>

> On Apr 29, 2020, at 10:08, Ning, Gang <[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.
> *****
>
> Dear all,
>
> We are in process of acquiring a 2P microscope. One of the applications is to cut zebrafish axon while imaging. We failed to cut nerves with a demo system equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300 nm plus a fixed line at 1045 nm). The attempt was done with a 25x water dipping lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep. I have to say the demo test was done in a relative short time on a new system which we don't know much. My questions are -
>
>  *   Any of you there have done 2p ablation with young/adult fish nerve? If so what equipment, laser, and parameters did you use to cut?
>  *   Any of you there have done 2p ablation with a water dipping lens without coverslip?
>  *   Any opinion about the laser for ablation, Spectra Physics vs Coherent?
>
> Thank you very much in advance, and stay safe and healthy!
>
> Greg
>
>
> Gang (Greg) Ning
>
> Microscopy Facility
>
> Huck Institutes of the Life Sciences
>
> Penn State University
>
> MSC N-048
>
> University Park, PA 16802
>
> 814-863-0994
>
> https://www.huck.psu.edu/core-facilities/microscopy-facility

[Christian Wilms]

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*****

While the MicroPoint is indeed a nice system, I think it will not be suitable for what Greg and this users are trying to achieve for two reasons:

1) Reaching over 100 um depth (750 um is what Greg mentioned) with a 440 nm laser to ablate an axon (and not everything above it) will be nigh impossible. NIR illumination and 2P ablation is required.
2) My understanding is that the MicroPoint will only allow for targeting of a spot using a camera image, acquired in the software controlling the MicroPoint - it would not allow a user to acquire an image with the two-photon microscope and load that image into the MicroPoint software.

In general, I can mostly echo what the Craig and Doug have said: Yes, it is possible (I have seen it done in a range of tissues). Water dipping should be fine. A wavelength longer than what you initially used should be more successful. Scan slowly - potentially just park the beam on your axon. Repeated fast scanning will not cause the same damage as a single slow scan - usually we want to do the former to avoid damage, in this case you want the latter to cause it.

And finally, yes, shorter pulses will be better. But keep in mind you will need to compress the pulses to the shortest possible duration under the objective. Ideally you would want to measure the pulse width under the objective with an autocorrelator (not cheap and takes a bit of time), or you can optimize the pulse duration while imaging using the wavelength you want to ablate with: change the GDD-compensation to get the brightest possible image (if you aren't doing that already).

Coherent vs. Spectra: either should work fine.

Best of luck,

Christian

Dr. Christian Wilms / Research & Development Manager
[hidden email] / [phone]
www.scientifica.uk.com

Take a look at our NeuroWire blog to see our latest news, guides, videos and more.

> -----Original Message-----
> From: Kalpana Iyengar <[hidden email]>
> Sent: 01 May 2020 18:04
> Subject: **Commercial Response* Re: 2P for zebrafish axon ablation
>
> *****
> 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.
> *****
>
> **Commercial Response**
>
> Hello Greg,
>
> I want to direct you to a couple of publications in which the researchers use the
> Andor Micropoint laser system for axon ablation at 440 nm:
> doi:10.1242/dev.004267
> doi:10.1038/nn1803
>
> We have an excellent photo-ablation system that has been very successful in
> similar experiments and can be fully automated. It can also be used in
> conjunction with any microscope system.
>
> If you would like to learn more, I invite you to schedule a time to chat with us
> here: https://doodle.com/poll/chqeceehnnu67un6
> <https://doodle.com/poll/chqeceehnnu67un6>
>
> Hope this informs. Stay safe!
>
> Best,
> Kalpana Iyengar, Ph.D.
> Confocal Applications Scientist
> Andor Technology
> [phone]
> [hidden email] <mailto:[hidden email]>
>
> > On Apr 29, 2020, at 10:08, Ning, Gang <[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.
> > *****
> >
> > Dear all,
> >
> > We are in process of acquiring a 2P microscope. One of the applications is to
> cut zebrafish axon while imaging. We failed to cut nerves with a demo system
> equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300 nm
> plus a fixed line at 1045 nm). The attempt was done with a 25x water dipping
> lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep. I have to
> say the demo test was done in a relative short time on a new system which we
> don't know much. My questions are -
> >
> >  *   Any of you there have done 2p ablation with young/adult fish nerve? If so
> what equipment, laser, and parameters did you use to cut?
> >  *   Any of you there have done 2p ablation with a water dipping lens without
> coverslip?
> >  *   Any opinion about the laser for ablation, Spectra Physics vs Coherent?
> >
> > Thank you very much in advance, and stay safe and healthy!
> >
> > Greg
> >
> >
> > Gang (Greg) Ning
> >
> > Microscopy Facility
> >
> > Huck Institutes of the Life Sciences
> >
> > Penn State University
> >
> > MSC N-048
> >
> > University Park, PA 16802
> >
> > 814-863-0994
> >
> > https://www.huck.psu.edu/core-facilities/microscopy-facility

[samuel connell]

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Good Day Everyone, Please Pardon a brief interruption in your normal
list-serv academic traffic. We are providing a live demo/webinar series
this week for those who want a deeper look at the SoRa Super Resolution
implementation. Details Below:

Cheers,
-
Sam Connell

We are hosting another day of webinars presenting a live demo of
3i's implementation of easy-to-use super-resolution microscopy with the
Yokogawa CSU-W1 SoRa on our live-cell imaging platforms.

CSU-W1 SoRa is an easy-to-use super-resolution microscopy solution
utilizing a dual Nipkow disk pair with microlenses on both the illuminating
and pinhole disks. The resulting raw images have a 1.4x resolution
improvement and with deconvolution one can achieve twice the resolution of
raw spinning disk data. The high-speed benefits of spinning disk confocal
are well established, with a maximum speed of 200fps, low photodamage
compared to other super-resolution methods, and no limitation on dyes or
fluors for sample labeling. SoRa is available for new systems as well as an
upgrade for existing CSU-W1 systems.

For more information on the CSU-W1 SoRa, visit
https://www.intelligent-imaging.com/spinning-disk-confocal
<https://www.intelligent-imaging.com/spinning-disk-confocal#SoRa>.

Please sign up at our Webinars
<https://www.intelligent-imaging.com/webinars#sora_may> page:

   - Session 1: May 5 | 3PM BST | 4PM CEST
   <https://us02web.zoom.us/webinar/register/WN_7CBxRUjlRRSPGaSnZtNsrg>
   - Session 2: May 5 | 10AM PDT | 11AM MDT | 1PM EDT
   <https://us02web.zoom.us/webinar/register/WN_dy8by3S4TM20o4AELFMbbg>
   - Session 3: May 6 | 10AM CST | 12PM AEST
   <https://us02web.zoom.us/webinar/register/WN_MN5YTxL_QIWyS6spN5LWSw>

We look forward to seeing you there!

ST May 5 - 10AM PDT | 11AM MDT | 1PM EDT May 6 - 10AM CST | 12AM AEST
http://intelligent-imaging.com/webinarspular demand, join us for another
#Webinar on easy-to-use #SuperResolution Spinning Disk Confocal (#SDC)
#Microscopy with @Yokogawa_LS CSU-W1 SoRa: May 5 - 3PM BST | 4PM CEST May 5
- 10AM PDT | 11AM MDT | 1PM EDT May 6 - 10AM CST | 12AM AEST http://
intelligent-imaging.com/webinars

[Craig Brideau]

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*****

A quick and dirty way to verify that you have near minimum pulse width is
to grow a KDP crystal on a cover slip and use it to generate second
harmonic at the focus of your objective. Just create a super-saturated
solution of KDP powder in boiling water, and put a drop on the coverslip.
The crystals should start to form as the solution dries. You can also add a
sprinkle of KDP powder to the drop to provide a seed and get better crystal
formation. Adjust your pulse compressor to achieve maximum second harmonic
production. This isn't perfect but will allow you to compensate for your
microscope optics, which will be a far larger contributor to pulse spread
than your sample.

Craig

On Mon, May 4, 2020 at 3:13 AM Christian Wilms <
[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.
> *****
>
> While the MicroPoint is indeed a nice system, I think it will not be
> suitable for what Greg and this users are trying to achieve for two reasons:
>
> 1) Reaching over 100 um depth (750 um is what Greg mentioned) with a 440
> nm laser to ablate an axon (and not everything above it) will be nigh
> impossible. NIR illumination and 2P ablation is required.
> 2) My understanding is that the MicroPoint will only allow for targeting
> of a spot using a camera image, acquired in the software controlling the
> MicroPoint - it would not allow a user to acquire an image with the
> two-photon microscope and load that image into the MicroPoint software.
>
> In general, I can mostly echo what the Craig and Doug have said: Yes, it
> is possible (I have seen it done in a range of tissues). Water dipping
> should be fine. A wavelength longer than what you initially used should be
> more successful. Scan slowly - potentially just park the beam on your axon.
> Repeated fast scanning will not cause the same damage as a single slow scan
> - usually we want to do the former to avoid damage, in this case you want
> the latter to cause it.
>
> And finally, yes, shorter pulses will be better. But keep in mind you will
> need to compress the pulses to the shortest possible duration under the
> objective. Ideally you would want to measure the pulse width under the
> objective with an autocorrelator (not cheap and takes a bit of time), or
> you can optimize the pulse duration while imaging using the wavelength you
> want to ablate with: change the GDD-compensation to get the brightest
> possible image (if you aren't doing that already).
>
> Coherent vs. Spectra: either should work fine.
>
> Best of luck,
>
> Christian
>
> Dr. Christian Wilms / Research & Development Manager
> [hidden email] / [phone]
> www.scientifica.uk.com
>
> Take a look at our NeuroWire blog to see our latest news, guides, videos
> and more.
>
> > -----Original Message-----
> > From: Kalpana Iyengar <[hidden email]>
> > Sent: 01 May 2020 18:04
> > Subject: **Commercial Response* Re: 2P for zebrafish axon ablation
> >
> > *****
> > 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.
> > *****
> >
> > **Commercial Response**
> >
> > Hello Greg,
> >
> > I want to direct you to a couple of publications in which the
> researchers use the
> > Andor Micropoint laser system for axon ablation at 440 nm:
> > doi:10.1242/dev.004267
> > doi:10.1038/nn1803
> >
> > We have an excellent photo-ablation system that has been very successful
> in
> > similar experiments and can be fully automated. It can also be used in
> > conjunction with any microscope system.
> >
> > If you would like to learn more, I invite you to schedule a time to chat
> with us
> > here: https://doodle.com/poll/chqeceehnnu67un6
> > <https://doodle.com/poll/chqeceehnnu67un6>
> >
> > Hope this informs. Stay safe!
> >
> > Best,
> > Kalpana Iyengar, Ph.D.
> > Confocal Applications Scientist
> > Andor Technology
> > [phone]
> > [hidden email] <mailto:[hidden email]>
> >
> > > On Apr 29, 2020, at 10:08, Ning, Gang <[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.
> > > *****
> > >
> > > Dear all,
> > >
> > > We are in process of acquiring a 2P microscope. One of the
> applications is to
> > cut zebrafish axon while imaging. We failed to cut nerves with a demo
> system
> > equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300 nm
> > plus a fixed line at 1045 nm). The attempt was done with a 25x water
> dipping
> > lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep. I
> have to
> > say the demo test was done in a relative short time on a new system
> which we
> > don't know much. My questions are -
> > >
> > >  *   Any of you there have done 2p ablation with young/adult fish
> nerve? If so
> > what equipment, laser, and parameters did you use to cut?
> > >  *   Any of you there have done 2p ablation with a water dipping lens
> without
> > coverslip?
> > >  *   Any opinion about the laser for ablation, Spectra Physics vs
> Coherent?
> > >
> > > Thank you very much in advance, and stay safe and healthy!
> > >
> > > Greg
> > >
> > >
> > > Gang (Greg) Ning
> > >
> > > Microscopy Facility
> > >
> > > Huck Institutes of the Life Sciences
> > >
> > > Penn State University
> > >
> > > MSC N-048
> > >
> > > University Park, PA 16802
> > >
> > > 814-863-0994
> > >
> > > https://www.huck.psu.edu/core-facilities/microscopy-facility
>

[Kalpana Iyengar]

*****
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*****

Hi Christian,

Thank you for your response. You are correct in that I underestimated the need for penetration depth in this specific application. The MicroPoint has good power at 626 nm, however, we have not tested the depth specifications. If Greg or anyone else here is interested in investigating the depth requirements further, we are happy to help look into this.

However, I want to clarify that your second point is not accurate. We have developed a Virtual Camera approach that is designed specifically to integrate with third-party systems like this. We are able to do exactly what you mention: acquire an image with the two-photon microscope and load that image into the MicroPoint software.

Best,
Kalpana Iyengar, Ph.D.
Confocal Applications Scientist
Andor Technology
[phone]
[hidden email] <mailto:[hidden email]>

> On May 4, 2020, at 05:12, Christian Wilms <[hidden email]> wrote:
>
> While the MicroPoint is indeed a nice system, I think it will not be suitable for what Greg and this users are trying to achieve for two reasons:
>
> 1) Reaching over 100 um depth (750 um is what Greg mentioned) with a 440 nm laser to ablate an axon (and not everything above it) will be nigh impossible. NIR illumination and 2P ablation is required.
> 2) My understanding is that the MicroPoint will only allow for targeting of a spot using a camera image, acquired in the software controlling the MicroPoint - it would not allow a user to acquire an image with the two-photon microscope and load that image into the MicroPoint software.
>
> In general, I can mostly echo what the Craig and Doug have said: Yes, it is possible (I have seen it done in a range of tissues). Water dipping should be fine. A wavelength longer than what you initially used should be more successful. Scan slowly - potentially just park the beam on your axon. Repeated fast scanning will not cause the same damage as a single slow scan - usually we want to do the former to avoid damage, in this case you want the latter to cause it.
>
> And finally, yes, shorter pulses will be better. But keep in mind you will need to compress the pulses to the shortest possible duration under the objective. Ideally you would want to measure the pulse width under the objective with an autocorrelator (not cheap and takes a bit of time), or you can optimize the pulse duration while imaging using the wavelength you want to ablate with: change the GDD-compensation to get the brightest possible image (if you aren't doing that already).
>
> Coherent vs. Spectra: either should work fine.
>
> Best of luck,
>
> Christian
>
> Dr. Christian Wilms / Research & Development Manager
> [hidden email] <mailto:[hidden email]> / [phone]
> www.scientifica.uk.com <http://www.scientifica.uk.com/>
>
> Take a look at our NeuroWire blog to see our latest news, guides, videos and more.
>
>> -----Original Message-----
>> From: Kalpana Iyengar <[hidden email] <mailto:[hidden email]>>
>> Sent: 01 May 2020 18:04
>> Subject: **Commercial Response* Re: 2P for zebrafish axon ablation
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy <http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> Post images on http://www.imgur.com <http://www.imgur.com/> and include the link in your posting.
>> *****
>>
>> **Commercial Response**
>>
>> Hello Greg,
>>
>> I want to direct you to a couple of publications in which the researchers use the
>> Andor Micropoint laser system for axon ablation at 440 nm:
>> doi:10.1242/dev.004267
>> doi:10.1038/nn1803
>>
>> We have an excellent photo-ablation system that has been very successful in
>> similar experiments and can be fully automated. It can also be used in
>> conjunction with any microscope system.
>>
>> If you would like to learn more, I invite you to schedule a time to chat with us
>> here: https://doodle.com/poll/chqeceehnnu67un6
>> <https://doodle.com/poll/chqeceehnnu67un6 <https://doodle.com/poll/chqeceehnnu67un6>>
>>
>> Hope this informs. Stay safe!
>>
>> Best,
>> Kalpana Iyengar, Ph.D.
>> Confocal Applications Scientist
>> Andor Technology
>> [phone]
>> [hidden email] <mailto:[hidden email]> <mailto:[hidden email] <mailto:[hidden email]>>
>>
>>> On Apr 29, 2020, at 10:08, Ning, Gang <[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.
>>> *****
>>>
>>> Dear all,
>>>
>>> We are in process of acquiring a 2P microscope. One of the applications is to
>> cut zebrafish axon while imaging. We failed to cut nerves with a demo system
>> equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300 nm
>> plus a fixed line at 1045 nm). The attempt was done with a 25x water dipping
>> lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep. I have to
>> say the demo test was done in a relative short time on a new system which we
>> don't know much. My questions are -
>>>
>>> *   Any of you there have done 2p ablation with young/adult fish nerve? If so
>> what equipment, laser, and parameters did you use to cut?
>>> *   Any of you there have done 2p ablation with a water dipping lens without
>> coverslip?
>>> *   Any opinion about the laser for ablation, Spectra Physics vs Coherent?
>>>
>>> Thank you very much in advance, and stay safe and healthy!
>>>
>>> Greg
>>>
>>>
>>> Gang (Greg) Ning
>>>
>>> Microscopy Facility
>>>
>>> Huck Institutes of the Life Sciences
>>>
>>> Penn State University
>>>
>>> MSC N-048
>>>
>>> University Park, PA 16802
>>>
>>> 814-863-0994
>>>
>>> https://www.huck.psu.edu/core-facilities/microscopy-facility

[Zdenek Svindrych-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.
*****

Hi Craig,
we normally used urea for this. It's easier to find in the lab...

But a more general and pressing question is: Is the ablation indeed a
two-photon process? If so, then I'd guess it would not be powerful enough
to cause anything. Pure absorption will be much stronger, and pulse
duration will not matter, as long as it's in the nanoseconds or less, to
prevent heat dissipation.
And as a note, I would be very surprised if any decent femtosecond laser
wasn't able to cut an axon. One can "boil" the sample if not cautious
enough, and some papers I've seen jut park the laser on the cell for one
second at 100 mW power... this has to work.
And if it still does not work, making the cell a little bit more absorbent
at the NIR wavelength will enhance the effect dramatically. I'm thinking of
something like Indocyanine Green (though I have no idea how to get it into
the cells, or at least close to the surface)...
Best, zdenek

On Mon, May 4, 2020 at 3:24 PM 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.
> *****
>
> A quick and dirty way to verify that you have near minimum pulse width is
> to grow a KDP crystal on a cover slip and use it to generate second
> harmonic at the focus of your objective. Just create a super-saturated
> solution of KDP powder in boiling water, and put a drop on the coverslip.
> The crystals should start to form as the solution dries. You can also add a
> sprinkle of KDP powder to the drop to provide a seed and get better crystal
> formation. Adjust your pulse compressor to achieve maximum second harmonic
> production. This isn't perfect but will allow you to compensate for your
> microscope optics, which will be a far larger contributor to pulse spread
> than your sample.
>
> Craig
>
> On Mon, May 4, 2020 at 3:13 AM Christian Wilms <
> [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.
> > *****
> >
> > While the MicroPoint is indeed a nice system, I think it will not be
> > suitable for what Greg and this users are trying to achieve for two
> reasons:
> >
> > 1) Reaching over 100 um depth (750 um is what Greg mentioned) with a 440
> > nm laser to ablate an axon (and not everything above it) will be nigh
> > impossible. NIR illumination and 2P ablation is required.
> > 2) My understanding is that the MicroPoint will only allow for targeting
> > of a spot using a camera image, acquired in the software controlling the
> > MicroPoint - it would not allow a user to acquire an image with the
> > two-photon microscope and load that image into the MicroPoint software.
> >
> > In general, I can mostly echo what the Craig and Doug have said: Yes, it
> > is possible (I have seen it done in a range of tissues). Water dipping
> > should be fine. A wavelength longer than what you initially used should
> be
> > more successful. Scan slowly - potentially just park the beam on your
> axon.
> > Repeated fast scanning will not cause the same damage as a single slow
> scan
> > - usually we want to do the former to avoid damage, in this case you want
> > the latter to cause it.
> >
> > And finally, yes, shorter pulses will be better. But keep in mind you
> will
> > need to compress the pulses to the shortest possible duration under the
> > objective. Ideally you would want to measure the pulse width under the
> > objective with an autocorrelator (not cheap and takes a bit of time), or
> > you can optimize the pulse duration while imaging using the wavelength
> you
> > want to ablate with: change the GDD-compensation to get the brightest
> > possible image (if you aren't doing that already).
> >
> > Coherent vs. Spectra: either should work fine.
> >
> > Best of luck,
> >
> > Christian
> >
> > Dr. Christian Wilms / Research & Development Manager
> > [hidden email] / [phone]
> > www.scientifica.uk.com
> >
> > Take a look at our NeuroWire blog to see our latest news, guides, videos
> > and more.
> >
> > > -----Original Message-----
> > > From: Kalpana Iyengar <[hidden email]>
> > > Sent: 01 May 2020 18:04
> > > Subject: **Commercial Response* Re: 2P for zebrafish axon ablation
> > >
> > > *****
> > > 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.
> > > *****
> > >
> > > **Commercial Response**
> > >
> > > Hello Greg,
> > >
> > > I want to direct you to a couple of publications in which the
> > researchers use the
> > > Andor Micropoint laser system for axon ablation at 440 nm:
> > > doi:10.1242/dev.004267
> > > doi:10.1038/nn1803
> > >
> > > We have an excellent photo-ablation system that has been very
> successful
> > in
> > > similar experiments and can be fully automated. It can also be used in
> > > conjunction with any microscope system.
> > >
> > > If you would like to learn more, I invite you to schedule a time to
> chat
> > with us
> > > here: https://doodle.com/poll/chqeceehnnu67un6
> > > <https://doodle.com/poll/chqeceehnnu67un6>
> > >
> > > Hope this informs. Stay safe!
> > >
> > > Best,
> > > Kalpana Iyengar, Ph.D.
> > > Confocal Applications Scientist
> > > Andor Technology
> > > [phone]
> > > [hidden email] <mailto:[hidden email]>
> > >
> > > > On Apr 29, 2020, at 10:08, Ning, Gang <[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.
> > > > *****
> > > >
> > > > Dear all,
> > > >
> > > > We are in process of acquiring a 2P microscope. One of the
> > applications is to
> > > cut zebrafish axon while imaging. We failed to cut nerves with a demo
> > system
> > > equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300
> nm
> > > plus a fixed line at 1045 nm). The attempt was done with a 25x water
> > dipping
> > > lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep.
> I
> > have to
> > > say the demo test was done in a relative short time on a new system
> > which we
> > > don't know much. My questions are -
> > > >
> > > >  *   Any of you there have done 2p ablation with young/adult fish
> > nerve? If so
> > > what equipment, laser, and parameters did you use to cut?
> > > >  *   Any of you there have done 2p ablation with a water dipping lens
> > without
> > > coverslip?
> > > >  *   Any opinion about the laser for ablation, Spectra Physics vs
> > Coherent?
> > > >
> > > > Thank you very much in advance, and stay safe and healthy!
> > > >
> > > > Greg
> > > >
> > > >
> > > > Gang (Greg) Ning
> > > >
> > > > Microscopy Facility
> > > >
> > > > Huck Institutes of the Life Sciences
> > > >
> > > > Penn State University
> > > >
> > > > MSC N-048
> > > >
> > > > University Park, PA 16802
> > > >
> > > > 814-863-0994
> > > >
> > > > https://www.huck.psu.edu/core-facilities/microscopy-facility
> >
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Christian Wilms]

*****
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.
*****

Thanks for that correction Kalpana! Last time I spoke to one of your colleagues in UK headquarters is nearly a year ago. At the time I was told that function was available and wasn’t planned – so I am glad to hear that has changed.

Thanks Craig for the advice on using SHG.

Dr. Christian Wilms / Research & Development Manager
[hidden email]<mailto:[hidden email]> / [phone]
www.scientifica.uk.com<http://www.scientifica.uk.com/>

Take a look at our NeuroWire<https://www.scientifica.uk.com/neurowire-blog> blog to see our latest news, guides, videos and more.

From: Kalpana Iyengar <[hidden email]>
Sent: 05 May 2020 01:34
To: Christian Wilms <[hidden email]>
Cc: Confocal Microscopy List <[hidden email]>
Subject: Re: **Commercial Response* Re: 2P for zebrafish axon ablation

Hi Christian,

Thank you for your response. You are correct in that I underestimated the need for penetration depth in this specific application. The MicroPoint has good power at 626 nm, however, we have not tested the depth specifications. If Greg or anyone else here is interested in investigating the depth requirements further, we are happy to help look into this.

However, I want to clarify that your second point is not accurate. We have developed a Virtual Camera approach that is designed specifically to integrate with third-party systems like this. We are able to do exactly what you mention: acquire an image with the two-photon microscope and load that image into the MicroPoint software.

Best,
Kalpana Iyengar, Ph.D.
Confocal Applications Scientist
Andor Technology
[phone]
[hidden email]<mailto:[hidden email]>


On May 4, 2020, at 05:12, Christian Wilms <[hidden email]<mailto:[hidden email]>> wrote:

While the MicroPoint is indeed a nice system, I think it will not be suitable for what Greg and this users are trying to achieve for two reasons:

1) Reaching over 100 um depth (750 um is what Greg mentioned) with a 440 nm laser to ablate an axon (and not everything above it) will be nigh impossible. NIR illumination and 2P ablation is required.
2) My understanding is that the MicroPoint will only allow for targeting of a spot using a camera image, acquired in the software controlling the MicroPoint - it would not allow a user to acquire an image with the two-photon microscope and load that image into the MicroPoint software.

In general, I can mostly echo what the Craig and Doug have said: Yes, it is possible (I have seen it done in a range of tissues). Water dipping should be fine. A wavelength longer than what you initially used should be more successful. Scan slowly - potentially just park the beam on your axon. Repeated fast scanning will not cause the same damage as a single slow scan - usually we want to do the former to avoid damage, in this case you want the latter to cause it.

And finally, yes, shorter pulses will be better. But keep in mind you will need to compress the pulses to the shortest possible duration under the objective. Ideally you would want to measure the pulse width under the objective with an autocorrelator (not cheap and takes a bit of time), or you can optimize the pulse duration while imaging using the wavelength you want to ablate with: change the GDD-compensation to get the brightest possible image (if you aren't doing that already).

Coherent vs. Spectra: either should work fine.

Best of luck,

Christian

Dr. Christian Wilms / Research & Development Manager
[hidden email]<mailto:[hidden email]> / [phone]
www.scientifica.uk.com<http://www.scientifica.uk.com/>

Take a look at our NeuroWire blog to see our latest news, guides, videos and more.


-----Original Message-----
From: Kalpana Iyengar <[hidden email]<mailto:[hidden email]>>
Sent: 01 May 2020 18:04
Subject: **Commercial Response* Re: 2P for zebrafish axon ablation

*****
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<http://www.imgur.com/> and include the link in your posting.
*****

**Commercial Response**

Hello Greg,

I want to direct you to a couple of publications in which the researchers use the
Andor Micropoint laser system for axon ablation at 440 nm:
doi:10.1242/dev.004267
doi:10.1038/nn1803

We have an excellent photo-ablation system that has been very successful in
similar experiments and can be fully automated. It can also be used in
conjunction with any microscope system.

If you would like to learn more, I invite you to schedule a time to chat with us
here: https://doodle.com/poll/chqeceehnnu67un6
<https://doodle.com/poll/chqeceehnnu67un6>

Hope this informs. Stay safe!

Best,
Kalpana Iyengar, Ph.D.
Confocal Applications Scientist
Andor Technology
[phone]
[hidden email]<mailto:[hidden email]> <mailto:[hidden email]>


On Apr 29, 2020, at 10:08, Ning, Gang <[hidden email]<mailto:[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.
*****

Dear all,

We are in process of acquiring a 2P microscope. One of the applications is to
cut zebrafish axon while imaging. We failed to cut nerves with a demo system
equipped with Spectra Physics Insight X3 DUAL (tunable from 680 – 1300 nm
plus a fixed line at 1045 nm). The attempt was done with a 25x water dipping
lens NA1.0 without coverslip on a 7-day-old fish at about 750 um deep. I have to
say the demo test was done in a relative short time on a new system which we
don't know much. My questions are -


*   Any of you there have done 2p ablation with young/adult fish nerve? If so
what equipment, laser, and parameters did you use to cut?

*   Any of you there have done 2p ablation with a water dipping lens without
coverslip?

*   Any opinion about the laser for ablation, Spectra Physics vs Coherent?

Thank you very much in advance, and stay safe and healthy!

Greg


Gang (Greg) Ning

Microscopy Facility

Huck Institutes of the Life Sciences

Penn State University

MSC N-048

University Park, PA 16802

814-863-0994

https://www.huck.psu.edu/core-facilities/microscopy-facility