Non-linear Optogenetics

Kumar, Amit (2014) Non-linear Optogenetics. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Optical stimulation of genetically-targeted cells has evolved as an effective tool for the study of cellular systems, both in-vitro and in-vivo. Unlike the conventional single-photon Optogenetic methods, Two-Photon Optogentic Stimulation (TPOS) uses near-infrared (NIR) laser beam and hence enables higher spatial precision and temporal precision. In addition, the TPOS technique developed by Dhakal et al. also allows deeper penetration due to low scattering and absorption coefficients of tissue in the NIR spectral region. It may be noted that until now the two-photon cross section of ChR2 has been estimated based on a two-state model. However, our experimental data somewhat contradict the theory presented by Tank et al. and we have a feeling that a reversible four-state model for 2-photon excitation will fit the results better. Hence, here, we report the development of a four-state model for TPOS of ChR2 and present detailed study of the photocurrent arising from the FO-TPOS of HEK cells. Moreover, it has been observed that the power dependence of the current was highly non-linear. We, therefore, hypothesise contribution of single-photon stimulation by second harmonic generation of ultrafast NIR laser in ATR in addition to the current induced by two-photon stimulation. We, thus, have outlined the contributions to the net photocurrent by several potential phenomena, like the Second HArmonic Generation (SHG) in all-trans retinal (ATR), capacitive charging of cell membrane by NIR laser, etc., that take place simultaneously during NIR stimulation.

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