People have been talking about doing this for many years, but this article is the first I’ve seen that describes a practical two-photon microscope that I’ve seen that can image a decent field of view (e.g., 150 microns x 150 microns x 150 microns) at “over 100 volumes per second, at the resolution limit.” And the whole thing — laser included — costs around $40,000. Paper shows sample images as well as schematics and protocols.
— posted by Ed
neurodudes 
This does look like a great system, but the one thing that really stands out is the laser’s wavelength output, which was on the high side and used to image YFP. I am not too knowledgeable about optics, but it wasn’t clear to me whether this system could be used to excite say OGB1, or if not whether the AODs described could be used with a Ti:Sapphire laser. Would appreciate clarification from someone more knowledgeable.
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The 1033nm wavelength is short enough to excite Oregon Green 488 (through two-photon absorption) but not very effectively. Oregon Green 514 and Calcium Green would be better choices given limited laser power; Calcium Orange would work too, it would get excited at the blue edge of the absorption peak. We’re happy with Calcium Green-1, pure salt and dextran-conjugated versions, electroporated.
Molecular Probes (now Invitrogen) has a very nice spectrum viewer at http://probes.invitrogen.com/servlets/spectraviewer?fileid1=3010ca where you can compare spectra of the various fluorophores. Anything with significant absorption near 516nm (one half of 1033nm) will fluoresce to some extent through two-photon absorption, although the conversion from absolute one-photon to two-photon cross-sections is not straightforward. (If you use very high powers or ridiculously short pulses then anything at all that has atoms in it will fluoresce through more-than-two-photon absorption, but then you’d have the exploding tissue problem; keep your pulses long enough to minimize three-photon absorption and your tissue will stay alive and happy for a long time.)
1033nm is also short enough to excite EGFP (again, not very effectively), so all the fluorescent proteins south of EGFP (YFP, citrine, mOrange, mCherry, dsRed, etc.) can be excited with the one laser at the same time. 1033nm is too long for CFP or the original wild-type GFP.
AODs are cut and aligned for one wavelength, so the one we bought would be poorly suited for 800nm but would work fine with a Ti:sapphire laser tuned to 1033nm. The three main problems with Ti:sapphire at such a long wavelength are that it costs about fifty times more than an Ytterbium laser, it’s difficult or impossible to get long pulses, and that there isn’t one for sale with such a low noise (quarter percent RMS at 10kHz!!!).
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