A Photostable Silicon Rhodamine Platform for Optical Voltage Sensing

Huang, Yi‐Lin; Walker, Abigail; Miller, Evan W.

doi:10.1021/jacs.5b06644

Cited by 217 publications

(290 citation statements)

References 58 publications

(123 reference statements)

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“…2C). As predicted, and as demonstrated for previous classes of PeT-based voltage sensors (23,24,26), depolarization results in fluorescence enhancement from the cell membrane whereas hyperpolarization decreases membrane fluorescence (Fig. 2C).…”

Section: Resultssupporting

confidence: 88%

“…Additionally, because VF dyes are small molecules, they should be readily tunable to a number of different chromophores (25) to select for desired properties, such as wavelength (26), improved photostability, or enhanced two-photon cross-section (27). In this article, we describe the design, synthesis, characterization, and application of Rhodol VoltageFluor-5 (RVF5), a voltage-sensitive small molecule in the VoltageFluor family with enhanced photostability and improved two-photon optical cross-section relative to the parent fluorescein-based VF dyes.…”

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confidence: 99%

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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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Section: Resultssupporting

confidence: 88%

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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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“…To date, very few reports have investigated the electrophysiological maturation of hPSC derived mDA neurons, and standard electrophysiology is a low throughput method that only enabled investigation of ~6 neurons per condition33. We recently developed a novel approach to optically measure voltage with fluorescent dyes with higher throughput3435, and by applying this method to ~100 D40 neurons (culture conditions depicted in Fig. 1c) we observed that 39% of cells generated in 2D exhibited action potentials (Fig.…”

Section: Resultsmentioning

confidence: 99%

Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform

Adil

Rodrigues

Kulkarni

et al. 2017

Sci Rep

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Pluripotent stem cells (PSCs) have major potential as an unlimited source of functional cells for many biomedical applications; however, the development of cell manufacturing systems to enable this promise faces many challenges. For example, there have been major recent advances in the generation of midbrain dopaminergic (mDA) neurons from stem cells for Parkinson’s Disease (PD) therapy; however, production of these cells typically involves undefined components and difficult to scale 2D culture formats. Here, we used a fully defined, 3D, thermoresponsive biomaterial platform to rapidly generate large numbers of action-potential firing mDA neurons after 25 days of differentiation (~40% tyrosine hydroxylase (TH) positive, maturing into 25% cells exhibiting mDA neuron-like spiking behavior). Importantly, mDA neurons generated in 3D exhibited a 30-fold increase in viability upon implantation into rat striatum compared to neurons generated on 2D, consistent with the elevated expression of survival markers FOXA2 and EN1 in 3D. A defined, scalable, and resource-efficient cell culture platform can thus rapidly generate high quality differentiated cells, both neurons and potentially other cell types, with strong potential to accelerate both basic and translational research.

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“…11 Given the success of far-red voltage dyes like BeRST 1 in integrating multiple functional signals simultaneously, we wondered whether we might be able to perform two-color imaging simultaneously with RhoVR 1, despite the tighter optical overlap between rhodamines and GFP-based chromophores. Expression of cytosolic GFP in HEK cells stained with RhoVR 1 did not result in significant bleed-through of GFP fluorescence into the rhodamine channel and did not substantially diminish the voltage sensitivity of RhoVR-1 in voltage-clamped HEK cells (47±3% without vs. 45±1% with GFP, SI Fig.…”

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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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A Photostable Silicon Rhodamine Platform for Optical Voltage Sensing

Cited by 217 publications

References 58 publications

Voltage-sensitive rhodol with enhanced two-photon brightness

Voltage-sensitive rhodol with enhanced two-photon brightness

Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform

Isomerically Pure Tetramethylrhodamine Voltage Reporters

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