Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation

Abstract

The use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1.

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

“…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). Similarly, the slower thermal decay of L-MAG0 460 suggests that this PTL should be relatively insensitive to the method of 2P stimulation.…”

“…Using digital holography (2P-DH) (13,34), we demonstrate that activation of L-MAG0 460 is compatible with functional imaging using the red genetically encoded calcium indicator R-GECO1.0 (35). This combination of optical tools facilitates fast, multicellular stimulation and recording, an important benchmark for the utility of PTLs for in vivo optogenetic studies.…”

All-Optical Interrogation of Neural Circuits

“…After propagation through the objective, the beam is focused onto an intensity pattern, reproducing the desired template. Several recent reports implemented computergenerated holography for neuronal activation via one-photon and two-photon glutamate uncaging (Lutz et al, 2008;Nikolenko et al, 2008;Dal Maschio et al, 2010;Zahid et al, 2010;Anselmi et al, 2011;Yang et al, 2011) and actuation of opsin-expressing neurons in vitro and in vivo (Packer et al, 2012(Packer et al, , 2015Bègue et al, 2013;. CGH can also generate threedimensional multipoint illumination patterns, a feature demonstrated to perform multitrap optical tweezing (Curtis et al, 2002) and 3D glutamate uncaging Go et al, 2012).…”

“…A more useful all-optical strategy might involve blue-light-driven inhibitors alongside R-CaMP2, although such a combination has not yet been reported. Particularly interesting in this regard might be the new blue-light-driven inhibitory chloride channels, dubbed anion channel rhodopsins (Berndt et al, 2014;Wietek et al, 2014;Govorunova et al, 2015), integrated with R-CaMP2 under one-or two-photon control.…”

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures

“…35 In addition, temporally focused shapes show high robustness to scattering, further recommending this approach for extended shape fluorescence excitation in depth. 36,37 One undisputable disadvantage of CGH is the inhomogeneous light distribution within generated light patterns. These intensity "speckles" vary by 15% to 20% for one-photon excitation, and up to ∼50% for two-photon, due to the quadratic dependence of the signal on the excitation density.…”