Two-photon excitation of channelrhodopsin-2 at saturation

Abstract

We demonstrate that channelrhodopsin-2 (CR), a light-gated ion channel that is conventionally activated by using visible-light excitation, can also be activated by using IR two-photon excitation (TPE). An empirical estimate of CR's two-photon absorption crosssection at λ = 920 nm is presented, with a value (260 ± 20 GM) indicating that TPE stimulation of CR photocurrents is not typically limited by intrinsic molecular excitability [1 GM = 10 −50 (cm 4 s)/ photon]. By using direct physiological measurements of CR photocurrents and a model of ground-state depletion, we evaluate how saturation of CR's current-conducting state influences the spatial resolution of focused TPE photostimulation, and how photocurrents stimulated by using low-power scanning TPE temporally summate. We show that TPE, like visible-light excitation, can be used to stimulate action potentials in cultured CR-expressing neurons.optogenetics | photostimulation | laser-scanning microscopy C hannelrhodopsin-2 (CR) is a microbially derived cation channel that transiently opens in response to blue-light illumination (1). Several modified forms of the channel, each encoded by a single gene, have been expressed heterologously in excitable mammalian neurons to recapitulate this light-gated conductance. It has been shown that in CR-expressing neurons, action potentials can be triggered by single-photon excitation, using blue-light illumination, with millisecond temporal resolution (2-7). Where multiple cells are photosensitized, the optical resolution with which each cell can be independently excited determines the precision of optical neuronal interrogation.Spatially localized excitation of individual cells is, however, difficult to achieve in thick biological tissue by using single-photon excitation. Brain tissue, for example, is an optically scattering medium that defocuses light, reducing the spatial definition and intensity of a focused beam or projected spatial pattern with increasing tissue depth (8). Additionally, excitation cannot be confined to a single z-focal plane of interest perpendicular to the axis of illumination (but see ref. 9), a geometry typically favored for in vivo studies of neural circuits.Two-photon laser-scanning microscopy (TPLSM) using a focused IR laser beam is the method of choice by which to achieve spatially localized fluorescence excitation deep in scattering tissue, providing an intrinsic optical section around the plane of focus (10, 11). We hypothesized that an analogous method to activate CR photocurrents by using two-photon excitation (TPE) would confer a much higher degree of spatial precision for targeted neuronal photostimulation than is currently possible by using single-photon (blue-light) illumination.Here we use whole-cell recordings of photocurrents in cultured cells to provide an initial biophysical characterization of TPE of CR. At typical light intensities used for TPLSM, we find that brief (<25 ms) CR photocurrents are described by a singlephotocycle model incorporating depletion of the CR ground state....

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

“…Because MAGs can be used for controlling neuron depolarization when tethered to an engineered ion channel (e.g., LiGluR), we note that the 2P absorption cross-sections are smaller than that of Channelrhodopsin2 (230-260 GM) (12,39). Although it can be difficult to compare σ 2 values measured by different methods (16,17), Yamaguchi and Tahara reported a comparable σ 2 of 230 GM for all-trans retinal in solution determined by the same 2P spectrophotometry technique used here (39).…”

“…However, L-MAG0 460 offer some flexibility in 2P excitation compared with opsins because of their relatively slow off-kinetics (52,53). Two-photon stimulation of opsins with fast off-kinetics has required creative use of scanning algorithms (12) or scanless imaging methods (54) to achieve sufficient summation. Opsins that are specifically engineered to have slower off-kinetics can be excited by 2P laser scanning (11), although larger currents still are obtained by 2P-DH (34).…”

“…Brightness, defined for fluorophores as the product of absorption cross-section and fluorescence quantum yield, gives the experimenter an objective metric to assess fluorescent reporters and identify appropriate optical parameters, such as the optimal excitation wavelength and range of light intensities (18). By comparison, relatively little information is available on the 2P properties of optogenetic and photopharmacological tools (12,14). Wider adoption of 2P optical manipulations should follow from quantitative characterization of 2P brightness for optogenetic and photopharmacological reagents.…”

“…In particular, the inherent spatial confinement of 2P absorption is critical for optical manipulation of individual cells (10) in the intact mammalian brain, where it is difficult to control the spatial extent of gene expression or confine soluble reagents. However, 2P-excited optogenetics is not yet as widespread in adoption as 2PM, being widely perceived to require sophisticated optical techniques (11)(12)(13) or reagent concentrations that compromise pharmacological specificity (2,14).…”

ZnO nanotube waveguide arrays on graphene films for local optical excitation on biological cells

“…Single molecule fluorescence in biological systems requires highly localized optical excitation. 8,9 Several techniques have been used to reduce the excitation volume, including laser focusing, 10,11 two photon excitation, 12,13 4Pi, 14 image interference and incoherent interference illumination microscopy (I5M), 15 and near field excitation. 16,17 However, most of these methods involve complex and bulky optical systems.…”