An interesting paper on two-photon activation of channelrhodopsin-2 is out in Biophysical Journal. In In-depth activation of ChR2 sensitized excitable cells with high spatial resolution using two-photon excitation with near-IR laser microbeam, Mohanty et. al show cellular activation with a fast-scanning two-photon laser.
Action potential generation from Channelrhodopsin-2 with a two-photon beam has been difficult to achieve, presumably due to the small activation volume of the 2p spot. They show similar calcium transients in response to 2p stimulation as with one-photon stimulation. As depth increases, the one-photon response attenuates faster than the two-photon. Unfortunately, the supplemental info with electrophysiology traces are not yet online. Presumably, they are generating action potentials, but I’d like to see the raw data. Interestingly, they also show calcium increases when the laser stays in once place. This would imply that local depolarization causes local voltage-gated calcium channels to open, or that calcium is getting through the ChR2. I was under the impression that ChR2 has a low conductance for calcium, though this study by Caldwell et. al, in press for JBC, uses ChR2 specifically for its calcium permeability.
I’m not sure what to make of the first paper. Are they really able to fire action potentials with two-photon stimulation, at depth? Or are the calcium traces they are seeing simply the result of localized calcium flux. I’ll followup once the Supplemental Data becomes available. Still worth a look if this is the sort of thing you are interested in.