NetNotes


heat nanoparticles within cells, but I guess this does not affect the cal- culations! Xavier Sanjuan Samarra xavier.sanjuan@upf.edu


2-Photon Use for Zebrafish Axon Ablation Confocal Listserver 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). Te attempt was done with a 25×water dipping lens NA1.0 without coverslip on a 7-day-old fish at about 750 μm 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: Have any of you done 2P ablation with young/adult fish nerve? If so what equipment, laser, and parameters did you use to cut? Have any used a water dipping lens without coverslip? Any opinions about the laser used for ablation, Spectra Physics vs Coherent? Tank you very much in advance. Gang (Greg) Ning gxn7@psu.edu


You don’t mention what wavelength you were using. Have you


tried using deuterium for dipping? It’s less absorptive than water. Marc Reinig mreinig2@gmail.com


Te laser wavelength was 780 nm. Gang (Greg) Ning gxn7@psu.edu We’ve ablated cells in zebrafish with an Insight DS (the predeces-


sor to the X3 with less power), a 20×1.0 water dipping objective, wave- lengths 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 (80 fs vs. up to 200 fs) improves ablation ability. Douglas Rich- ardson ds.richardson@gmail.com


About 10 years ago I ablated focal lesions in spinal cord with an


old Spectra Physics Tsunami, which is pretty similar to contempo- rary Ti:Saph lasers. I believe the wavelength I used was 900 nm, and I dispersion-optimized the system for approximately 200 fs pulse width and used 10’s of mW on the sample. My dwell time was fairly long, I believe on the order of 50–250 microseconds. As Doug mentioned, shorter pulse widths will improve efficacy, so consider a pulse compres- sor. Craig Brideau craig.brideau@gmail.com


**Commercial Response** I want to direct you to a couple of


publications in which the researchers used the Andor Micropoint laser system for axon ablation at 440 nm: doi:10.1242/dev.004267 and 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. Kalpana Iyengar k.iyengar@andor.com


While the MicroPoint is indeed a nice system, I think it will


not be suitable for what Greg and his users are trying to achieve for two reasons: 1) Reaching over 100 μm depth (750 μm 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 soſtware controlling the MicroPoint. Tis would not allow a user to acquire an image with the two-photon microscope and load that image into the MicroPoint soſtware. In general, I can mostly echo what 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


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just park the beam on the 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. Christian Wilms christian.wilms@scientifica.uk.com


Hi Christian, Tank you for your response. You are correct in that


I underestimated the need for penetration depth in this specific appli- cation. Te MicroPoint has good power at 626 nm, however, we have not tested the depth specifications. If Greg or anyone else here is inter- ested 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 soſtware. Kalpana Iyengar k.iyengar@andor.com


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 gener- ate 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. Te 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. Tis 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 Brideau craig.brideau@gmail.com


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 just 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 ef- fect 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). Zdenek Svindrych zdedenn@gmail.com


Tanks for that correction, Kalpana! Last time I spoke to one of


your colleagues in the UK headquarters was nearly a year ago. At the time I was told that function was not available and wasn’t planned – so I am glad to hear that has changed. Tanks, Craig for the advice on us- ing SHG. Christian Wilms christian.wilms@scientifica.uk.com


Carbon Coater Microscopy Listserver We are seeking to purchase a new carbon coater. Our center does EDS and EBSD and we would like to coat down to 5 nm of carbon. We


www.microscopy-today.com • 2020 July


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