RESUMEN
Two-photon voltage imaging has long been heralded as a transformative approach capable of answering many long-standing questions in modern neuroscience. However, exploiting its full potential requires the development of novel imaging approaches well suited to the photophysical properties of genetically encoded voltage indicators. We demonstrate that parallel excitation approaches developed for scanless two-photon photostimulation enable high-SNR two-photon voltage imaging. We use whole-cell patch-clamp electrophysiology to perform a thorough characterization of scanless two-photon voltage imaging using three parallel illumination approaches and lasers with different repetition rates and wavelengths. We demonstrate voltage recordings of high-frequency spike trains and sub-threshold depolarizations from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-Kv. Using a low repetition-rate laser, we perform multi-cell recordings from up to fifteen targets simultaneously. We co-express JEDI-2P-Kv and the channelrhodopsin ChroME-ST and capitalize on their overlapping two-photon absorption spectra to simultaneously evoke and image action potentials using a single laser source. We also demonstrate in vivo scanless two-photon imaging of multiple cells simultaneously up to 250 µm deep in the barrel cortex of head-fixed, anaesthetised mice.
Asunto(s)
Potenciales de Acción , Neuronas , Fotones , Animales , Ratones , Neuronas/fisiología , Potenciales de Acción/fisiología , Técnicas de Placa-Clamp , Rayos LáserRESUMEN
Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs µm-2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs µm-2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs µm-2) in dense cytosolic tubulin datasets.
Asunto(s)
Imagenología Tridimensional , Microscopía , Microscopía/métodos , Imagenología Tridimensional/métodos , Imagen Individual de Molécula/métodos , NanotecnologíaRESUMEN
Parallel light-sculpting methods have been used to perform scanless two-photon photostimulation of multiple neurons simultaneously during all-optical neurophysiology experiments. We demonstrate that scanless two-photon excitation also enables high-resolution, high-contrast, voltage imaging by efficiently exciting fluorescence in a large fraction of the cellular soma. We present a thorough characterisation of scanless two-photon voltage imaging using existing parallel approaches and lasers with different repetition rates. We demonstrate voltage recordings of high frequency spike trains and sub-threshold depolarizations in intact brain tissue from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-kv. Using a low repetition-rate laser, we perform recordings from up to ten neurons simultaneously. Finally, by co-expressing JEDI-2P-kv and the channelrhodopsin ChroME-ST in neurons of hippocampal organotypic slices, we perform single-beam, simultaneous, two-photon voltage imaging and photostimulation. This enables in-situ validation of the precise number and timing of light evoked action potentials and will pave the way for rapid and scalable identification of functional brain connections in intact neural circuits.
RESUMEN
Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms.