Moulding, Dale |
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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 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 |
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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 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: > ***** > 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 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. > |
In reply to this post by Moulding, Dale
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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, 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 |
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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 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: > ***** > 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, > > 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 |
In reply to this post by Moulding, Dale
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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 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 |
In reply to this post by Moulding, Dale
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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 |
David Schoppik |
In reply to this post by Moulding, Dale
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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 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) |
In reply to this post by Moulding, Dale
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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 |
Craig Brideau |
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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. ***** 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 |
In reply to this post by Moulding, Dale
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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 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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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. ***** 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 ***** 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 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 ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
David Schoppik |
In reply to this post by Moulding, Dale
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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 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, 10151047 (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. |
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