Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca²⁺) concentration are powerful tools for visualizing intracellular signaling ...activity. However, despite a decade of availability, the palette of single FP-based Ca²⁺ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca²⁺ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca²⁺ was imaged in three subcellular compartments, and, in conjunction with a cyan FP—yellow FP—based indicator, Ca²⁺ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca²⁺ imaging.
Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new ...platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the ...brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
Understanding how the brain encodes and processes information requires the recording of neural activity that underlies different behaviors. Recent efforts in fluorescent protein engineering have ...succeeded in developing powerful tools for visualizing neural activity, in general by coupling neural activity to different properties of a fluorescent protein scaffold. Here, we take advantage of a previously unexploited class of reversibly switchable fluorescent proteins to engineer a new type of calcium sensor. We introduce rsCaMPARI, a genetically encoded calcium marker engineered from a reversibly switchable fluorescent protein that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely swimming zebrafish.
Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We ...developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca
) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, ...designated NIR-GECO1, enables imaging of Ca
transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca
imaging in combination with other optogenetic indicators and actuators.
Glutamate is one of the 20 common amino acids and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators ...for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development of noncircularly permutated (ncp) variants. R- and Rncp-iGluSnFR1 display glutamate affinities of 11 μM and 0.9 μM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of HEK-293 cells. Furthermore, we show that Gncp-iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons.
The synthesis of benzosuberone-fused heterocyclic derivatives is an increasingly intriguing subject. The current study reports the synthesis of the new ring system ...benzo6,7cyclohepta1,2-bthieno3,2-epyridine. At first, new 2-thioxo-2,5,6,7-tetrahydro-1H-benzo6,7cyclohepta1,2-bpyridine-3-carbonitriles were prepared and taken as versatile precursors for the present study. Afterward, a succession of α-bromoketones were combined with the newly generated synthons in ethanol that also included two equivalents of sodium ethoxide. After 2-3 h of heating at reflux, a novel series of benzo6,7cyclohepta1,2-bthieno3,2-epyridines was obtained in 86-93% yields. Using a similar protocol, a novel series of benzosuberone-fused bis(thieno3,2-epyridines) linked to different spacers was obtained. Therefore, two pyridine-2(1H)-thiones equivalents were reacted with one equivalent of the respective bis(α-bromoketones) in ethanol containing four equivalents of sodium ethoxide. The reaction mixture was refluxed for 4-6 h to produce the corresponding bis(benzo6,7cyclohepta1,2-bthieno3,2-epyridines) in 67-76% yields. Both spectral data and elemental analyses were used to elucidate the structure of new products.
Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high ...spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.
Abstract
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we ...present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.
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