Point ablation with Mutliphoton laser

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Moulding, Dale Moulding, Dale
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Point ablation with Mutliphoton laser

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Dear list,

I'd be very grateful for some advice regarding a multiphoton laser for point ablations. We
want to ablate individual centrosomes in zebrafish embryos without damaging the rest of
the cell. We plan to do this with water dipping objectives (NA ~1.0) on a confocal system
with an 80 MHz multiphoton laser.
What factors should we consider in the choice of laser?
Is there a benefit for extremely short pulse width (say ~75 fs vs ~140 fs)?
What range of average power or peak power should we be aiming for?
Are there any published reports or guidance for optimising ablations?

Many thanks

Dale

Imaging Facility Manager
UCL Institute of Child Health.
Craig Brideau Craig Brideau
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Re: Point ablation with Mutliphoton laser

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Hi Dale, the shorter the pulse you use, the less collateral damage there
will be. The shorter the pulse is in time, the broader its spectrum must
be. The broader its spectrum is, the harder it is to focus accurately due
to NIR chromatic aberration. The Coherent Vitara has adjustable bandwidth,
so you might be able to use that to play with the pulse parameters until
you get optimal ablation. I believe its center wavelength is fixed around
800nm approximately, but then you can alter the bandwidth around this
point. You would also need a pulse compression setup (Coherent of course
sells these too) to keep everything from getting excessively dispersed.
Another thing to consider is that high pulse energy can cause photochemical
damage, especially in the presence of oxygen, so this might be an issue for
you as well.

You are opening a can of worms with this one, but you could get some
interesting results!

Craig Brideau

On Tue, Jun 30, 2015 at 10:40 AM, Dale Moulding <[hidden email]>
wrote:

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> *****
>
> Dear list,
>
> I'd be very grateful for some advice regarding a multiphoton laser for
> point ablations. We
> want to ablate individual centrosomes in zebrafish embryos without
> damaging the rest of
> the cell. We plan to do this with water dipping objectives (NA ~1.0) on a
> confocal system
> with an 80 MHz multiphoton laser.
> What factors should we consider in the choice of laser?
> Is there a benefit for extremely short pulse width (say ~75 fs vs ~140 fs)?
> What range of average power or peak power should we be aiming for?
> Are there any published reports or guidance for optimising ablations?
>
> Many thanks
>
> Dale
>
> Imaging Facility Manager
> UCL Institute of Child Health.
>
weber weber
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Re: Point ablation with Mutliphoton laser

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Hi,

In fact you also have consider optical elements that are in the path.
But if you want a quick removal switch your choice will be on an AOM. Turn fast
to generate short pulses. The AOM is a crystal and he will disperse most
passing 100fs over 500fs. You can use a prechirp for dispersion.
The 20X, NA = 1.0 is a good choice. If you want to control the peak power you
must know the average power under the lens. (Thorlabs sells power-meter) to
measure the width of implusion you must use an autocorrelator. He alone will
allow you to edit the values of your system: APE berlin.
http://www.ape-berlin.de/sites/default/files/Carpe_0.pdf
http://www.ape-berlin.de

Also you must be sure to fill the rear element of the lens and check the
consistency of your field. For that I use a flat mirror and a visible laser to
ensure the setting.

With a lens 10 NA = 0.5 , I obtain with the zoom mini 5% of in-homogeneity.
(diagonals of the square)
for your bleaching it is always best to use the laser where it is most powerful.
Generally 800nm
Craig Brideau Craig Brideau
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Re: Point ablation with Mutliphoton laser

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Hi Pascal, rather than an AOM, I'd suggest a pulse picker for the laser.
It's a fairly common option for these types of systems. They let you adjust
the repetition rate of the laser to control the number of pulses hitting
the target within a given interval. For ablation this tends to be more
important than average power control. Higher end pulse pickers also act as
a shutter, allowing you to let through a short burst of pulses at a time
and rate, which is optimal for point ablation.

Craig Brideau


On Tue, Jun 30, 2015 at 11:29 AM, Pascal Weber <[hidden email]>
wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> Hi,
>
> In fact you also have consider optical elements that are in the path.
> But if you want a quick removal switch your choice will be on an AOM. Turn
> fast
> to generate short pulses. The AOM is a crystal and he will disperse most
> passing 100fs over 500fs. You can use a prechirp for dispersion.
> The 20X, NA = 1.0 is a good choice. If you want to control the peak power
> you
> must know the average power under the lens. (Thorlabs sells power-meter) to
> measure the width of implusion you must use an autocorrelator. He alone
> will
> allow you to edit the values of your system: APE berlin.
> http://www.ape-berlin.de/sites/default/files/Carpe_0.pdf
> http://www.ape-berlin.de
>
> Also you must be sure to fill the rear element of the lens and check the
> consistency of your field. For that I use a flat mirror and a visible
> laser to
> ensure the setting.
>
> With a lens 10 NA = 0.5 , I obtain with the zoom mini 5% of in-homogeneity.
> (diagonals of the square)
> for your bleaching it is always best to use the laser where it is most
> powerful.
> Generally 800nm
>
Reto Fiolka Reto Fiolka
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Re: Point ablation with Mutliphoton laser

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Hi Dale

While I agree with Craig and Pascal that with shorter laser pulses you have to be more
careful about dispersion, I do not think that you need the autocorrelator that Pascal
mentioned.

Since a prism compressor can only compensate GVD, it is sufficient to just optimize the
signal you get from a dye solution under 2 photon excitation under your microscope while
tuning the compressor.

I wonder though how the APE device works, is that a nonlinear autocorrelator? It is tough to
analyze a laser pulse just by its autocorrelation, as you do not have the spectral phase
information and many different pulse profiles can have the same autocorrelation. To really
diagnose dispersion, you would need the spectral phase, which to my knowledge only FROG
and SPIDER can give you for ultrafast pulses (commercially available, at least).

Best,
Reto
Moulding, Dale Moulding, Dale
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Re: Point ablation with Mutliphoton laser

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Dear all,

thanks for the very helpful replies. Clearly this is going to be an enjoyable and tricky
project. We are tied in to getting a 80 MHz tunable (~700nm to ~1040nm) laser, this will be
routed through an AOM. We need it for a multi-user facility mostly for deeper imaging of
cleared tissues. The point ablations project I expect to be the toughest to crack.
My choice really lies in whether to go for a ~70 fs or ~140 fs laser. These will have
dispersion / pre-chirp, but I am not sure whether the shorter pulse may prove more difficult
to focus on a very small region for point ablation? I plan to optimise by looking at bleaching
in similar tissues to calibrate the compensation.
So it boils down to whether the shorter 70 fs pulse (with broader spectrum) will outperform
the 140 fs pulse?

cheers

Dale
David Schoppik David Schoppik
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Re: Point ablation with Mutliphoton laser

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Hi Dale,
While we never attempted anything as small as organelles, my postdoc lab (Florian
Engert, Harvard University) had considerable experience ablating single cells in the
larval zebrafish brain. The cells I ablated were 150-200um deep from pigment-less
anesthetized fish that were 4-5dpf.

The original protocol was described in Orger et. al. Nat. Neurosci 2008. A slightly
updated version is in our paper, with controls to try and evaluate possible damage
to surrounding neurons: Bianco et. al. Curr. Biology 2012. Most recently, for some
unpublished (yet) work, we used a SpectraPhysics Mai Tai HP at peak
(~820nm), without any pre-chirping. Power was 200mW at the sample through an
Olympus 20x/1.0 objective. In all of our ablations rather than providing power for
a particular time/number of pulses, we monitored the PMT looking for a signature
saturating event and gating (mechanical shutter) the beam when observed.
Empirically, that worked surprisingly well as an indicator of cell ablation without
damage to surrounding tissue. As always, the devil is in the details.

The most elegant ablation work I know of in zebrafish comes from a collaboration
between the Schaffer and Fetcho labs at Cornell. They have targeted the dendrite
of the Mauthner cell (Shum et. al. 2008 Frontiers in Optics). Still not centrosome
small, but getting closer! They used femtosecond lasers that deliver low pulse
rates (~1kHz) at high power (~10uJ) and apply single pulses to great effect. The
reported pulse power was 50nJ / 100fs / 1kHz in that paper (power levels
achievable with the MaiTai HP). I believe the laser that they used
was one of these http://www.imra.com/products/product-lines/fcpa-microjewel/ 
which aren't inexpensive, but there are other manufacturers that have similar
products e.g. http://www.polaronyxlaser.com/products.php or
http://www.fianium.com/high-energy.htm that might work as well.

Good luck!
Cole, Richard W (HEALTH) Cole, Richard W (HEALTH)
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Re: Point ablation with Mutliphoton laser

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Dale,

First off, if you are not committed to a multi-photon laser I would recommend a Nd:YAG laser.  These pulsed laser work extremely well for ablation studies.  I have personally built and used Nd:YAG rigs for cutting chromosome and ablating centrosomes.  MP laser are a bit trickery, and as other have mentioned you need pulse modulation.

Best of luck



Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
 
Research Associate Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York

P.O. Box 509 Albany N.Y. 12201-0509
518-474-7048 Phone
518-408-1730 Fax

Website www.wadsworth.org/cores/alm/index.htm
Craig Brideau Craig Brideau
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Re: Point ablation with Mutliphoton laser

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I worry that an Nd:YAG would cause too much peripheral damage. For the size
of target being discussed here a pulse laser will probably be required. I
still feel a short pulse laser in the 10s of fs and a good pulse picker
will be necessary.

Craig Brideau
On Jul 1, 2015 5:42 AM, "Cole, Richard (HEALTH)" <[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.
> *****
>
> Dale,
>
> First off, if you are not committed to a multi-photon laser I would
> recommend a Nd:YAG laser.  These pulsed laser work extremely well for
> ablation studies.  I have personally built and used Nd:YAG rigs for cutting
> chromosome and ablating centrosomes.  MP laser are a bit trickery, and as
> other have mentioned you need pulse modulation.
>
> Best of luck
>
>
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy & Image Analysis Core
> Wadsworth Center
>
> Research Associate Professor
> Dept. of Biomedical Sciences
> School of Public Health State University of New York
>
> P.O. Box 509 Albany N.Y. 12201-0509
> 518-474-7048 Phone
> 518-408-1730 Fax
>
> Website www.wadsworth.org/cores/alm/index.htm
>
Nuno Moreno Nuno Moreno
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Re: Point ablation with Mutliphoton laser

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*****
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Dear Dale

Just to reinforce David comment. I have tried with drosophila dorsal closer like 10 years ago in an MRC1024 MP. I remember that the tricky part was to figure out the right amount of energy before too much damage. Since everything was manual plus the non linear effect of the absorption and having no AOM, it turned out to be impossible with current settings. But it was superficial,  we just went back to pulsed UV.

I believe this problem will come first than 140 vs 70 fs

btw, and this is just speculation. UV will ionise tissues by braking chemical bounds. In 2p absorption, special if no adaptive optics is used to reshape the psf in deep ablation, heat would most likely be the predominant effect. I think this was discussed already in the confocal mailing list.

Best,
Nuno Moreno

> On 01 Jul 2015, at 10:28, Dale Moulding <[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,
>
> thanks for the very helpful replies. Clearly this is going to be an enjoyable and tricky
> project. We are tied in to getting a 80 MHz tunable (~700nm to ~1040nm) laser, this will be
> routed through an AOM. We need it for a multi-user facility mostly for deeper imaging of
> cleared tissues. The point ablations project I expect to be the toughest to crack.
> My choice really lies in whether to go for a ~70 fs or ~140 fs laser. These will have
> dispersion / pre-chirp, but I am not sure whether the shorter pulse may prove more difficult
> to focus on a very small region for point ablation? I plan to optimise by looking at bleaching
> in similar tissues to calibrate the compensation.
> So it boils down to whether the shorter 70 fs pulse (with broader spectrum) will outperform
> the 140 fs pulse?
>
> cheers
>
> Dale
mcammer mcammer
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Re: Point ablation with Mutliphoton laser

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Perhaps I am simply not adept enough at this, but in my hands either I get nothing or just a bit more power or dwell time blows a big hole.  Have tried from around 820 to 960 nm at 80 MHz.

But I  have seen success with photoconversion or photoactivaton of UV through blue sensitive probes.


_________________________________________
Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy
http://microscopynotes.com/
Cell: (914) 309-3270

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Nuno Moreno [[hidden email]]
Sent: Wednesday, July 01, 2015 11:53 AM
To: [hidden email]
Subject: Re: Point ablation with Mutliphoton laser

*****
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Dear Dale

Just to reinforce David comment. I have tried with drosophila dorsal closer like 10 years ago in an MRC1024 MP. I remember that the tricky part was to figure out the right amount of energy before too much damage. Since everything was manual plus the non linear effect of the absorption and having no AOM, it turned out to be impossible with current settings. But it was superficial,  we just went back to pulsed UV.

I believe this problem will come first than 140 vs 70 fs

btw, and this is just speculation. UV will ionise tissues by braking chemical bounds. In 2p absorption, special if no adaptive optics is used to reshape the psf in deep ablation, heat would most likely be the predominant effect. I think this was discussed already in the confocal mailing list.

Best,
Nuno Moreno

> On 01 Jul 2015, at 10:28, Dale Moulding <[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,
>
> thanks for the very helpful replies. Clearly this is going to be an enjoyable and tricky
> project. We are tied in to getting a 80 MHz tunable (~700nm to ~1040nm) laser, this will be
> routed through an AOM. We need it for a multi-user facility mostly for deeper imaging of
> cleared tissues. The point ablations project I expect to be the toughest to crack.
> My choice really lies in whether to go for a ~70 fs or ~140 fs laser. These will have
> dispersion / pre-chirp, but I am not sure whether the shorter pulse may prove more difficult
> to focus on a very small region for point ablation? I plan to optimise by looking at bleaching
> in similar tissues to calibrate the compensation.
> So it boils down to whether the shorter 70 fs pulse (with broader spectrum) will outperform
> the 140 fs pulse?
>
> cheers
>
> Dale

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Re: Point ablation with Mutliphoton laser

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Hi Michael & co.
Your experience sounds about right. Empirically, it's a fine line between ablation
and disaster, particularly if you don't have a way to gate the laser. On the plus
side, it's pretty clear when you've gone too far!

IIRC, the paper from the Schaffer/Fetcho group that I cited only has successful
ablations ~50% of the time. The remainder of the time was either cell death
(40%) or nothing (10%). Mechanistically, the best explanation of what's going on
that I've read is Vogel, Noack, Hüttman & Paltauf "Mechanisms of femtosecond
laser nanosurgery of cells and tissues" Appl. Phys. B 81, 1015–1047 (2005), esp.
Section 7. Would be curious to know of other references.

Maybe most useful for OP is the chapter by Tsai & Kleinfeld: "In Vivo Two-Photon
Laser Scanning Microscopy with Concurrent Plasma-Mediated Ablation Principles
and Hardware Realization"
https://physics.ucsd.edu/neurophysics/publications/CRC_chapter_3.pdf
There's a lot of useful information & theory in there.