Scanless two-photon excitation of channelrhodopsin-2

Wavefront-Engineering Microscopy Group, Neurophysiology and New Microscopies Laboratory, Centre National de Recherche Scientifique, Unité Mixte de Recherche 8154, Institut National de Santé et de Recherche Médicale U603, Paris Descartes University, Paris, France.
Nature Methods (Impact Factor: 32.07). 10/2010; 7(10):848-54. DOI: 10.1038/nmeth.1505
Source: PubMed


Light-gated ion channels and pumps have made it possible to probe intact neural circuits by manipulating the activity of groups of genetically similar neurons. What is needed now is a method for precisely aiming the stimulating light at single neuronal processes, neurons or groups of neurons. We developed a method that combines generalized phase contrast with temporal focusing (TF-GPC) to shape two-photon excitation for this purpose. The illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups of cells. The TF-GPC two-photon excitation patterns generated large photocurrents in Channelrhodopsin-2-expressing cultured cells and neurons and in mouse acute cortical slices. The amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and, thereby, be used to trigger single action potentials or trains of action potentials.

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    • "The GPC method provides a straightforward phase to intensity mapping using a simple 4f imaging setup and can be considered as a generalization of Zernike's phase contrast microscopy technique applied to beam shaping and optical information processing [11]. GPC has been used to generate speckle-free extended light patterns and has recently been combined with temporal focusing in rapidly reconfigurable two-photon optogenetics to create neuron-shaped excitations [12]. Prior theoretical and numerical predictions has been carried out to optimize and match GPC light shaping for Gaussian laser profiles [13] [14]. "
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    ABSTRACT: The Generalized Phase Contrast (GPC) method has been demonstrated to reshape light efficiently to match the input beam profile requirement of different illumination targets. A spatially coherent beam can be GPC-shaped into a variety of static and dynamic profiles to match e.g. fixed commercially available modulation systems or for more irregular and dynamic shapes such as found in advanced optogenetic light-excitations of neurons. In this work, we integrate a static GPC light shaper to illuminate a phase-only spatial light modulator encoding dynamic phase holograms. The GPC-enhanced phase-holograms are encoded to create reconfigurable spot arrays and arbitrary extended patterns. For a given laser power, our experimental results show a significant intensity gain in the resulting diffraction patterns when we illuminate the holograms with a GPC-shaped beam as compared to the more common practice of hard truncation. The phase flatness of the GPC-enhanced readout beam has also been investigated.
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    • "Further, the initial phase of the illumination pattern must be found prior to the image reconstruction process. In digital light processing techniques, the use of a spatial light modulator (SLM) and digital micromirror device (DMD) have been proposed to generate arbitrary patterns in tandem with initial phase control, different phase shift, and rotation angle of illumination patterns for fast optical switching [29], pattern excitation in optogenetics [6], and high-throughput microfabrication [30]. Consequently, the second-order NSIM with projected structured light can be delivered via a DMD; and further, the conventional diffraction grating can be replaced by a DMD in TFMPEM [31]. "
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    ABSTRACT: In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.
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    • "focal spots of laser illumination [147], [148]. While the latter study yielded effective stimulation of single cells, the amount of laser power required to generate action potentials was quite high (>100 mW). "
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    ABSTRACT: The brain is a large network of interconnected neurons where each cell functions as a nonlinear processing element. Unraveling the mysteries of information processing in the complex networks of the brain requires versatile neurostimulation and imaging techniques. Optogenetics is a new stimulation method which allows the activity of neurons to be modulated by light. For this purpose, the cell-types of interest are genetically targeted to produce light-sensitive proteins. Once these proteins are expressed, neural activity can be controlled by exposing the cells to light of appropriate wavelengths. Optogenetics provides a unique combination of features, including multimodal control over neural function and genetic targeting of specific cell-types. Together, these versatile features combine to a powerful experimental approach, suitable for the study of the circuitry of psychiatric and neurological disorders. The advent of optogenetics was followed by extensive research aimed to produce new lines of light-sensitive proteins and to develop new technologies: for example, to control the distribution of light inside the brain tissue or to combine optogenetics with other modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance imaging. In this paper, the authors review some of the recent advances in the field of optogenetics and related technologies and provide their vision for the future of the field.
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