Non-linear stimulation of excitable cells with and without optogenetic sensitization

Shivaranjani Shivalingaiah; Ling Gu; Samarendra K. Mohanty

doi:10.1117/12.875996

18 February 2011 Non-linear stimulation of excitable cells with and without optogenetic sensitization

Shivaranjani Shivalingaiah, Ling Gu, Samarendra K. Mohanty

Proceedings Volume 7883, Photonic Therapeutics and Diagnostics VII; 788355 (2011) https://doi.org/10.1117/12.875996
Event: SPIE BiOS, 2011, San Francisco, California, United States

Abstract

Here, we report development of an integrated system for co-registration of patch-clamp measurements with calcium imaging during two-photon stimulation (TPS) of excitable cells sensitized with optogenetic probe, chanelrhodopsin-2 (ChR2). Comparison of calcium changes due to focused two-photon micro-irradiation of excitable cells with and without optogenetic sensitization, revealed wavelength-insensitive injection of extra-cellular calcium via pore formation at high laser beam powers. However, use of defocused/weakly-focused beam allowed sub-threshold stimulation of the excitable cells, revealed by both calcium imaging and whole-cell patch-clamping. Irregular calcium spiking was observed for continuous two-photon defocused micro-irradiation. Even at high extra-cellular calcium conditions, since presence of alltrans- retinal (ATR) was necessary even for detectable calcium increase (and inward current) under defocused twophoton irradiation, role of ChR2 was confirmed as opposed to optoporation, for defocused condition. In the subthreshold stimulation regime, while peak-photocurrents variation with TPS wavelength followed ChR2 two-photon activation spectrum, the power dependence of the current was highly non-linear. Though defocused two-photon beam may cause minimal photo damage while stimulating the cells, the threshold average power required for generating action potential in the ChR2-sensitized cells is higher than that used for routine two-photon imaging.

Citation Download Citation

Shivaranjani Shivalingaiah, Ling Gu, and Samarendra K. Mohanty "Non-linear stimulation of excitable cells with and without optogenetic sensitization", Proc. SPIE 7883, Photonic Therapeutics and Diagnostics VII, 788355 (18 February 2011); https://doi.org/10.1117/12.875996

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