title: In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using two-photon excitation with a near-infrared laser microbeam.
abstract: We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.
authors.affiliation: Beckman Laser Institute, University of California, Irvine, California, USA. smohanty@uci.edu
authors.forename: Samarendra K
authors.initials: SK
authors.lastname: Mohanty
authors.forename: Rainer K
authors.initials: RK
authors.lastname: Reinscheid
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Liu
authors.forename: Naoe
authors.initials: N
authors.lastname: Okamura
authors.forename: Tatiana B
authors.initials: TB
authors.lastname: Krasieva
authors.forename: Michael W
authors.initials: MW
authors.lastname: Berns
mesh_terms: D000818:Animals; D002460:Cell Line; D000075402:Channelrhodopsins; D005453:Fluorescence; D006801:Humans; D007259:Infrared Rays; D015640:Ion Channel Gating / Q000528:radiation effects*; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D009930:Organic Chemicals / Q000378:metabolism; D017785:Photons
chemical_list: D000075402:Channelrhodopsins; D009930:Organic Chemicals; C071335:calcium orange