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A chemogenetic approach for optical monitoring of voltage in neurons
摘要： Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for interrogation of the cellular and molecular logic of the nervous system. Here we describe a Semisynthetic Tethered Voltage Indicator (STeVI1) based upon Nile Red that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and subthreshold activity in neurons.
关键词： membrane potential probes,voltage imaging,genetic targeting,protein tags,fluorogenic probes
更新于2025-09-23 15:22:29
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Strong pH-Dependent Near-Infrared Fluorescence in a Microbial Rhodopsin Reconstituted with a Red-Shifting Retinal Analogue
摘要： Near-infrared (NIR)-driven rhodopsins are of great interest in optogenetics and other optobiotechnological developments such as artificial photosynthesis and deep-tissue voltage imaging. Here we report the proton pump proteorhodopsin (PR) containing a NIR-active retinal analogue (PR:MMAR) exhibits intense NIR fluorescence at a quantum yield of 3.3%. This is 130 times higher than native PR (Lenz, M. O.; et al. Biophys J. 2006, 91, 255?262) and 3?8 times higher than the QuasAr and PROPS voltage sensors (Kralj, J.; et al. Science 2011, 333, 345?348; Hochbaum, D. R.; et al. Nat. Methods 2014, 11, 825?833). The NIR fluorescence strongly depends on the pH in the range of 6?8.5, suggesting potential application of MMAR-binding proteins as ultrasensitive NIR-driven pH and/or voltage sensors. Femtosecond transient absorption spectroscopy showed that upon near-IR excitation, PR:MMAR features an unusually long fluorescence lifetime of 310 ps and the absence of isomerized photoproducts, consistent with the high fluorescence quantum yield. Stimulated Raman analysis indicates that the NIR-absorbing species develops upon protonation of a conserved aspartate, which promotes charge delocalization and bond length leveling due to an additional methylamino group in MMAR, in essence providing a secondary protonated Schiff base. This results in much smaller bond length alteration along the conjugated backbone, thereby conferring significant single-bond character to the C13C14 bond and structural deformation of the chromophore, which interferes with photoinduced isomerization and extends the lifetime for fluorescence. Hence, our studies allow for a molecular understanding of the relation between absorption/emission wavelength, isomerization, and fluorescence in PR:MMAR. As acidification enhances the resonance state, this explains the strong pH dependence of the NIR emission.
关键词： stimulated Raman analysis,fluorescence,voltage sensor,rhodopsins,optogenetics,artificial photosynthesis,proteorhodopsin,femtosecond transient absorption spectroscopy,pH sensor,Near-infrared,voltage imaging
更新于2025-09-23 15:21:01
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A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
摘要： A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that is an order of magnitude brighter than FlicR1 at a resting potential of ?70 mV and exhibits a ten-fold larger change in fluorescence (ΔF) upon 100 mV depolarization of the plasma membrane in HEK 293 cells. Unlike ArcLight, the introduction of charged residues to the exterior of dTomato did not substantially improve the dynamic range of the optical signal. As a result, this new GEVI, Ilmol, yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1 despite a smaller fractional change in fluorescence of 4% per 100 mV depolarization of the plasma membrane. Ilmol expresses well in neurons resolving action potentials in neuronal cultures and reporting population signals in mouse hippocampal acute brain slice recordings. Ilmol is the brightest red-shifted GEVI to date enabling imaging with 160-fold less light than Archon1 for primary neuron recordings (50 mW/cm2 versus 8 W/cm2) and 600-fold less light than QuasAr2 for mouse brain slice recordings (500 mW/cm2 versus 300 W/cm2). This new GEVI uses a distinct mechanism from other approaches, opening an alternate engineering path to improve sensitivity and speed.
关键词： fluorescence,Genetically Encoded Voltage Indicator,tdTomato,optical signal,GEVI,neuronal activity,red-shifted,voltage imaging
更新于2025-09-23 15:21:01
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Autonomous Scan Patterns for Laser Voltage Imaging
摘要： The semiconductor industry ramping up design capabilities for emerging technologies is facing new test quality and yield management challenges. To facilitate debugging of the first silicon, diagnosis of yield issues, and to enable repair processes, an accurate defect isolation is needed with support of advanced test, diagnostic, and yield analysis tools. Laser voltage imaging (LVI), laser voltage probing (LVP), and electron-beam testing are popular diagnostic techniques for scan chain failures. As scan remains instrumental in developing more advanced DFT technologies, its reliable operations are essential for test pattern bring-up, failure analysis, and yield learning. Running LVI tests at early stages of a design process involves a sophisticated infrastructure to offer a nanometer diagnostic resolution with a non-destructive failure isolation. However, as the use of external testers may not be feasible besides providing power and clock signals, the paper demonstrates how to reuse on-chip EDT compression environment to generate and apply LVI-aware scan patterns for advanced contactless test procedures. The presented approach avoids the repetitive loading of scan chains, requires no seed patterns to encode LVI tests, has virtually no area overhead, and offers a flexible selection of non-toggling scan chains in a low power test mode. Furthermore, there are no constraints or requirements imposed by designs that could limit the applicability of the scheme. The proposed solution does not compromise the test compression performance, marked by test data reduction ratios, test coverage, and test time. Hence, there are no modifications needed in existing flows and tools to implement the method besides the use of recommended polynomials.
关键词： scan cell diagnosis,logic BIST,repeated scan integrity tests,test compression,E-beam test,laser voltage imaging
更新于2025-09-16 10:30:52