scholarly journals Population imaging discrepancies between a genetically-encoded calcium indicator (GECI) versus a genetically-encoded voltage indicator (GEVI)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei Hong Zhu ◽  
Jinyoung Jang ◽  
Milena M. Milosevic ◽  
Srdjan D. Antic
Keyword(s):  
Brain Slices ◽  
Imaging Modalities ◽  
Population Activity ◽  
Optical Signals ◽  
Voltage Imaging ◽  
Neuronal Populations ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
Genetically Encoded Calcium Indicators ◽  
Voltage Indicator

AbstractGenetically-encoded calcium indicators (GECIs) are essential for studying brain function, while voltage indicators (GEVIs) are slowly permeating neuroscience. Fundamentally, GECI and GEVI measure different things, but both are advertised as reporters of “neuronal activity”. We quantified the similarities and differences between calcium and voltage imaging modalities, in the context of population activity (without single-cell resolution) in brain slices. GECI optical signals showed 8–20 times better SNR than GEVI signals, but GECI signals attenuated more with distance from the stimulation site. We show the exact temporal discrepancy between calcium and voltage imaging modalities, and discuss the misleading aspects of GECI imaging. For example, population voltage signals already repolarized to the baseline (~ disappeared), while the GECI signals were still near maximum. The region-to-region propagation latencies, easily captured by GEVI imaging, are blurred in GECI imaging. Temporal summation of GECI signals is highly exaggerated, causing uniform voltage events produced by neuronal populations to appear with highly variable amplitudes in GECI population traces. Relative signal amplitudes in GECI recordings are thus misleading. In simultaneous recordings from multiple sites, the compound EPSP signals in cortical neuropil (population signals) are less distorted by GEVIs than by GECIs.

scholarly journals Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

2021 ◽  
Author(s):  
Ahmed S Abdelfattah ◽  
Jihong Zheng ◽  
Daniel Reep ◽  
Getahun Tsegaye ◽  
Arthur Tsang ◽  
...  
Keyword(s):  
Saturation Mutagenesis ◽  
Voltage Sensitivity ◽  
Voltage Imaging ◽  
Neuronal Populations ◽  
Lower Baseline ◽  
Transmembrane Voltage ◽  
Automated Patch Clamp ◽  
Subthreshold Voltage ◽  
Voltage Indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

scholarly journals FIOLA: An accelerated pipeline for Fluorescence Imaging OnLine Analysis

2021 ◽  
Author(s):  
Andrea Giovannucci ◽  
Changjia Cai ◽  
Cynthia Dong ◽  
Marton Rozsa ◽  
Eftychios Pnevmatikakis
Keyword(s):  
Real Time ◽  
Fluorescence Imaging ◽  
Neural Activity ◽  
Simulated Data ◽  
Real Data ◽  
Imaging Modalities ◽  
Imaging Data ◽  
Online Analysis ◽  
Voltage Imaging ◽  
Neuronal Populations

Abstract Optical microscopy methods such as calcium and voltage imaging already enable fast activity readout (30-1000Hz) of large neuronal populations using light. However, the lack of corresponding advances in online algorithms has slowed progress in retrieving information about neural activity during or shortly after an experiment. This technological gap not only prevents the execution of novel real-time closed-loop experiments, but also hampers fast experiment-analysis-theory turnover for high-throughput imaging modalities. The fundamental challenge is to reliably extract neural activity from fluorescence imaging frames at speeds compatible with new indicator dynamics and imaging modalities. To meet these challenges and requirements, we propose a framework for Fluorescence Imaging OnLine Analysis (FIOLA). FIOLA exploits computational graphs and accelerated hardware to preprocess fluorescence imaging movies and extract fluorescence traces at speeds in excess of 300Hz on calcium imaging datasets and at speeds over 400Hz on voltage imaging datasets. Besides, we present the first real-time spike extraction algorithm for voltage imaging data. We evaluate FIOLA on both simulated data and real data, demonstrating reliable and scalable performance. Our methods provide the computational substrate required to interface precisely large neuronal populations and machines in real-time, enabling new applications in neuroprosthetics, brain-machine interfaces, and experimental neuroscience. Moreover, this new set of tools is poised to dramatically shorten the experiment-data-theory cycle by providing immediate feedback on the activity of large neuronal populations at experimental time.

scholarly journals High-speed, multi-Z confocal microscopy for voltage imaging in densely labeled neuronal populations

2021 ◽  
Author(s):  
Timothy D Weber ◽  
Maria V Moya ◽  
Jerome Mertz ◽  
Michael N Economo
Keyword(s):  
High Speed ◽  
Signal To Noise Ratio ◽  
Neuronal Population ◽  
Great Promise ◽  
High Signal ◽  
Signal To Noise ◽  
Population Activity ◽  
Voltage Imaging ◽  
Neuronal Populations ◽  
Optical Crosstalk

Genetically encoded voltage indicators (GEVIs) hold great promise for monitoring neuronal population activity, but GEVI imaging in dense neuronal populations remains difficult due to a lack of contrast and/or speed. To address this challenge, we developed a novel confocal microscope that allows simultaneous multiplane imaging with high-contrast at near-kHz rates. This approach enables high signal-to-noise ratio voltage imaging in densely labeled populations and minimizes optical crosstalk during concurrent optogenetic photostimulation.

scholarly journals Blind sparse deconvolution for inferring spike trains from fluorescence recordings

2017 ◽  
Author(s):  
Jérôme Tubiana ◽  
Sébastien Wolf ◽  
Georges Debregeas
Keyword(s):  
Action Potentials ◽  
Imaging Techniques ◽  
Real Data ◽  
Computational Framework ◽  
Systems Neuroscience ◽  
Neuronal Populations ◽  
Calcium Indicators ◽  
Genetically Encoded Calcium Indicators

The parallel developments of genetically-encoded calcium indicators and fast fluorescence imaging techniques makes it possible to simultaneously record neural activity of extended neuronal populations in vivo, opening a new arena for systems neuroscience. To fully harness the potential of functional imaging, one needs to infer the sequence of action potentials from fluorescence time traces. Here we build on recently proposed computational approaches to develop a blind sparse deconvolution algorithm (BSD), which we motivate by a theoretical analysis. We demonstrate that this method outperforms existing sparse deconvolution algorithms in terms of robustness, speed and/or accuracy on both synthetic and real fluorescence data. Furthermore, we provide solutions for the practical problems of thresholding and determination of the rise and decay time constants. We provide theoretical bounds on the performance of the algorithm in terms of precision-recall and temporal accuracy. Finally, we extend the computational framework to support temporal superresolution whose performance is established on real data.

scholarly journals A positively tuned voltage indicator reveals electrical correlates of calcium activity in the brain

2021 ◽  
Author(s):  
Stephen Wenceslao Evans ◽  
Dongqing Shi ◽  
Mariya Chavarha ◽  
Mark Houston Plitt ◽  
Jiannis Taxidis ◽  
...  
Keyword(s):  
Temporal Resolution ◽  
Calcium Imaging ◽  
Photon Detection ◽  
Temporal Precision ◽  
Voltage Imaging ◽  
Calcium Indicators ◽  
Subthreshold Voltage ◽  
Relationship Of ◽  
Genetically Encoded Calcium Indicators

Neuronal activity is routinely recorded in vivo using genetically encoded calcium indicators (GECIs) and 2-photon microscopy, but calcium imaging is poorly sensitive for single voltage spikes under typical population imaging conditions, lacks temporal precision, and does not report subthreshold voltage changes. Genetically encoded voltage indicators (GEVIs) offer better temporal resolution and subthreshold sensitivity, but 2-photon detection of single spikes in vivo using GEVIs has required specialized imaging equipment. Here, we report ASAP4b and ASAP4e, two GEVIs that brighten in response to membrane depolarization, inverting the fluorescence-voltage relationship of previous ASAP-family GEVIs. ASAP4b and ASAP4e feature 180% and 210% fluorescence increases to 100-mV depolarizations, respectively, as well as modestly prolonged deactivation and high photostability. We demonstrate single-trial detection of spikes and oscillations in vivo with standard 1 and 2-photon imaging systems, and confirm improved temporal resolution in comparison to calcium imaging on the same equipment. Thus, ASAP4b and ASAP4e GEVIs extend the uses of existing imaging equipment to include multi-unit voltage imaging in vivo.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

Genetically Encoded Calcium Indicators

2008 ◽  
Vol 108 (5) ◽  
pp. 1550-1564 ◽  
Author(s):  
Marco Mank ◽  
Oliver Griesbeck
Keyword(s):  
Calcium Indicators ◽  
Genetically Encoded Calcium Indicators

scholarly journals All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

2021 ◽  
Author(s):  
He Tian ◽  
Hunter C. Davis ◽  
J. David Wong-Campos ◽  
Linlin Z. Fan ◽  
Benjamin Gmeiner ◽  
...  
Keyword(s):  
Gap Junction ◽  
Signal To Noise Ratio ◽  
Voltage Imaging ◽  
All Optical ◽  
Precision Voltage ◽  
Coupling Strengths ◽  
Junction Coupling ◽  
Multiple Cells ◽  
Voltage Indicator

All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The "Optopatch" constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.

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