Small molecule fluorescent voltage indicators for studying membrane potential

Miller, Evan W.

doi:10.1016/j.cbpa.2016.06.003

Cited by 111 publications

(131 citation statements)

References 42 publications

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“…Direct imaging of voltage changes with fluorescent probes is an attractive solution to these challenges because voltage imaging can provide the spatial and temporal resolution needed to match signals to cells with high throughput and fidelity (2,3). The challenge of achieving optical voltage sensing is a longstanding goal within the scientific community (4,5), and recent approaches have included fluorinated styryl dyes (6), annulated hemicyanines (7,8) and cyanines (9), lipophilic anions (10,11), hybrid small-molecule/fluorescent protein probes (12,13), porphyrins (14), and nanoparticles (15,16).…”

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confidence: 99%

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using twophoton (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue. Neuronal membrane potential dynamics drive neurotransmitter release and are therefore responsible for the unique physiology associated with neurons at cellular, circuit, and organismal levels. Despite the central importance of proper neuronal firing to human health, an integrated understanding of neuronal activity in the context of larger brain circuits remains elusive, due in part to a lack of methods for interrogating membrane potential dynamics with sufficient spatial and temporal resolution.Traditional methods for monitoring membrane potential rely heavily on the use of invasive electrodes, through one of two methods. The first method, patch-clamp electrophysiology, uses a single electrode to make contact with or puncture a cell to record changes in membrane potential, sacrificing throughput and spatial resolution to achieve a comprehensive description of a single cellular electrophysiological profile. A second method uses multielectrode arrays (MEAs), in which patterned arrays of electrodes introduced to cells or tissues report on electrical changes. Spatial resolution of MEAs depends on the number and positioning of the electrodes within the array. Although throughput is improved relative to patch-clamp electrophysiology, the extracellularly recorded signals are typically less sensitive than whole-cell methods and can be an amalgamation of several cells, making deconvolution of recorded signals and precise correlation to specific cells difficult or impossible. Addi...

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confidence: 99%

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…3 Broadly applicable and sensitive optical methods to track V m would expand our capacity to disentangle the contributions V m makes to human health and disease. 4–6 …”

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confidence: 99%

Isomerically Pure Tetramethylrhodamine Voltage Reporters

et al. 2016

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We present the design, synthesis, and application of a new family of fluorescent voltage indicators based on isomerically pure tetramethylrhodamines. These new Rhodamine Voltage Reporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing, display excitation and emission profiles in the green to orange region of the visible spectrum, demonstrate high sensitivity to membrane potential changes (up to 47% ΔF/F per 100 mV), and employ a tertiary amide derived from sarcosine, which aids in membrane localization and simultaneously simplifies the synthetic route to the voltage sensors. The most sensitive of the RhoVR dyes, RhoVR 1, features a methoxy-substituted diethylaniline donor and phenylenevinylene molecular wire in the 5′-position of the rhodamine aryl ring, the highest voltage-sensitivity to date for red-shifted PeT-based voltage sensors, and is compatible with simultaneous imaging alongside GFP-based indicators. The discoveries that sarcosine-based tertiary amides in the context of molecular wire voltage indicators prevent dye internalization, along with the improved voltage sensitivity of 5′-substituted voltage indicators should be broadly applicable to other types of PeT-based voltage-sensitive fluorophores.

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“…Traditional methods to measure membrane potential rely on invasive electrodes, introduced via pipette or on micro or nano-arrays. Voltage imaging with fluorescent indicators, whether genetically-encoded(16) or chemically synthesized,(15) is an attractive solution because such imaging circumvents problems of low-throughput, low spatial resolution, and high invasiveness associated with more traditional electrode-based techniques. (17–19) Our lab is developing VoltageFluor, or VF, dyes which are a small molecule platform for voltage imaging operating via a photoinduced electron transfer (PeT) quenching mechanism(20, 21) to directly image transmembrane voltage changes.…”

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confidence: 99%

A Rationally Designed, General Strategy for Membrane Orientation of Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes

Kulkarni¹,

Yin

Pourmandi³

et al. 2016

ACS Chem. Biol.

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Voltage imaging with fluorescent dyes offers promise for interrogating the complex roles of membrane potential in coordinating the activity of neurons in the brain. Yet, low sensitivity often limits the broad applicability of optical voltage indicators. In this paper, we use molecular dynamics (MD) simulations to guide the design of new, ultra-sensitive fluorescent voltage indicators that use photoinduced electron transfer (PeT) as a voltage-sensing switch. MD simulations predict an approximately 16% increase in voltage sensitivity resulting purely from improved alignment of dye with the membrane. We confirm this theoretical finding by synthesizing 9 new voltage-sensitive (VoltageFluor, or VF) dyes and establishing that all of them display the expected improvement of approximately 19%. This synergistic outworking of theory and experiment enabled computational and theoretical estimation of VF dye orientation in lipid bilayers and has yielded the most sensitive PeT-based VF dye to date. We use this new voltage indicator to monitor voltage spikes in neurons from rat hippocampus and human pluripotent stem cell-derived dopaminergic neurons.

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Small molecule fluorescent voltage indicators for studying membrane potential

Cited by 111 publications

References 42 publications

Voltage-sensitive rhodol with enhanced two-photon brightness

Voltage-sensitive rhodol with enhanced two-photon brightness

Isomerically Pure Tetramethylrhodamine Voltage Reporters

A Rationally Designed, General Strategy for Membrane Orientation of Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes

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