Improved PeT Molecules for Optically Sensing Voltage in Neurons

Woodford, Clifford Rodereigh; Frady, E. Paxon; Smith, Richard; Morey, Benjamin; Canzi, Gabriele; Palida, Sakina F.; Araneda, Ricardo C.; Kristan, William B.; Kubiak, Clifford P.; Miller, Evan W.; Tsien, Roger Y.

doi:10.1021/ja510602z

Cited by 111 publications

(164 citation statements)

References 42 publications

Supporting

Mentioning

145

Contrasting

Unclassified

Order By: Relevance

“…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: 89%

See 1 more Smart Citation

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

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

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Resultssupporting

confidence: 89%

“…1) (24). PeT within a VF dye is enhanced under hyperpolarized cellular conditions (negative intracellular charge) and decreased during cellular depolarization (positive intracellular charge).…”

mentioning

confidence: 99%

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…Functionalizing the ND surface with charged molecules or polymers could provide a mechanism for such charge transfer. For example, it has been shown that electrical activity in cells can be detected by the use of voltage-dependent charge transfer from molecular wires to a fluorescent reporter (31,32). Functionalizing hydrogenated NDs with similar molecular wires could provide the necessary charge movement and allow their use as voltage indicators.…”

Section: Resultsmentioning

confidence: 99%

Modulation of nitrogen vacancy charge state and fluorescence in nanodiamonds using electrochemical potential

Karaveli

Gaathon

Wolcott

et al. 2016

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

View full text Add to dashboard Cite

The negatively charged nitrogen vacancy (NV − ) center in diamond has attracted strong interest for a wide range of sensing and quantum information processing applications. To this end, recent work has focused on controlling the NV charge state, whose stability strongly depends on its electrostatic environment. Here, we demonstrate that the charge state and fluorescence dynamics of single NV centers in nanodiamonds with different surface terminations can be controlled by an externally applied potential difference in an electrochemical cell. The voltage dependence of the NV charge state can be used to stabilize the NV − state for spin-based sensing protocols and provides a method of charge state-dependent fluorescence sensing of electrochemical potentials. We detect clear NV fluorescence modulation for voltage changes down to 100 mV, with a single NV and down to 20 mV with multiple NV centers in a wide-field imaging mode. These results suggest that NV centers in nanodiamonds could enable parallel optical detection of biologically relevant electrochemical potentials. (Fig. 1A). The negative charge state (NV − ) is optically addressable and has a long-lived electronic spin state suitable for quantum sensing of local electric and magnetic fields (1-4). However, under constant laser illumination, the NV center can stochastically switch between the negatively charged state and the neutral charge state, NV 0 (5, 6). Because the NV 0 state lacks the NV − state's favorable spin properties, there has been efforts to engineer stable NV − centers in diamond devices (7). Studies have shown that the diamond surface termination strongly affects the charge state of NVs near the surface: hydrogen surface termination increases the fraction of NV 0 over NV −

show abstract

“…By combining this optical system with a new-generation voltage-sensitive dye (VSD), VoltageFluor (Miller et al ., 2012; Woodford et al ., 2015), fluorescence signals that encode membrane voltages with high fidelity can be simultaneously captured from neurons at different depths. We applied this pan-neuronal recording system to the nervous system of the medicinal leech, in which we elicited fictive behaviors and quantitatively manipulated membrane potential of identifiable neurons using electrophysiological methods (Tomina and Wagenaar, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…These procedures were not explained in detail in our previous paper (Tomina and Wagenaar, 2017), nor in other studies using the same type of dyes (Miller et al ., 2012, Moshtagh-Khorasani et al ., 2013, Woodford et al ., 2015, Frady et al ., 2016). …”

Section: Introductionmentioning

confidence: 99%

Dual-sided Voltage-sensitive Dye Imaging of Leech Ganglia

Tomina

Wagenaar

2018

BIO-PROTOCOL

View full text Add to dashboard Cite

In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems.

show abstract

Improved PeT Molecules for Optically Sensing Voltage in Neurons

Cited by 111 publications

References 42 publications

Voltage-sensitive rhodol with enhanced two-photon brightness

Voltage-sensitive rhodol with enhanced two-photon brightness

Modulation of nitrogen vacancy charge state and fluorescence in nanodiamonds using electrochemical potential

Dual-sided Voltage-sensitive Dye Imaging of Leech Ganglia

Contact Info

Product

Resources

About