Anellated Hemicyanine Dyes with Large Symmetrical Solvatochromism of Absorption and Fluorescence

Hübener, Gerd; Lambacher, Armin; Fromherz, Peter

doi:10.1021/jp0345809

Cited by 56 publications

(51 citation statements)

References 37 publications

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“…3). In certain trials, spike-evoked peak dF∕F approached values closer to theoretically estimated limits for electrochromic probes 7,29 [for example, 26% in Fig. 2(b)]…”

Section: Discussionsupporting

confidence: 75%

“…In conjunction with the rapidly expanding toolbox of organic 29,[40][41][42][43][44][45][46][47] and protein-based, genetically targetable voltage, [48][49][50][51][52][53][54][55][56][57] calcium, and neurotransmitter reporters, holographic light shaping can enable high S/N, parallel detection of neural-evoked fluorescence transients from neighboring cells or subcellular compartments. Here, we have demonstrated that light targeting increases spatial specificity even for cases in which the dye itself is confined to the membrane of a single cell.…”

Section: Discussionmentioning

confidence: 99%

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Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures

et al. 2015

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Abstract. Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large "pseudowidefield" (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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“…3). In certain trials, spike-evoked peak dF∕F approached values closer to theoretically estimated limits for electrochromic probes 7,29 [for example, 26% in Fig. 2(b)]…”

Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures

et al. 2015

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“…This small wavelength shift dictates that the fluorescence signal can be best recorded at the edges of the spectrum, where intensity varies most steeply as a function of wavelength. The largest linear responses are −28% ΔF/F per 100 mV (22), although more typical values are ∼10% per 100 mV (23,24). Photo-induced electron transfer (PeT)-based Ca 2+ probes, such as fluo-3, give ΔF/F values of up to 150% for action potentials in cultured hippocampal neurons (25).…”

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confidence: 99%

Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires

Miller¹,

Lin²,

Frady³

et al. 2012

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

284

323

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Fluorescence imaging is an attractive method for monitoring neuronal activity. A key challenge for optically monitoring voltage is development of sensors that can give large and fast responses to changes in transmembrane potential. We now present fluorescent sensors that detect voltage changes in neurons by modulation of photo-induced electron transfer (PeT) from an electron donor through a synthetic molecular wire to a fluorophore. These dyes give bigger responses to voltage than electrochromic dyes, yet have much faster kinetics and much less added capacitance than existing sensors based on hydrophobic anions or voltagesensitive ion channels. These features enable single-trial detection of synaptic and action potentials in cultured hippocampal neurons and intact leech ganglia. Voltage-dependent PeT should be amenable to much further optimization, but the existing probes are already valuable indicators of neuronal activity. F luorescence imaging can map the electrical activity and communication of multiple spatially resolved neurons and thus complements traditional electrophysiological measurements (1, 2). Ca 2+ imaging is the most popular of such techniques, because the indicators are well-developed (3-6), highly sensitive (5, 6), and genetically encodable (7-13), enabling investigation of the spatial distribution of Ca 2+ dynamics in structures as small as dendritic spines and as large as functional circuits. However, because neurons translate depolarizations into Ca 2+ signals via a complex series of pumps, channels, and buffers, fluorescence imaging of Ca 2+ transients cannot provide a complete picture of electrical activity in neurons. Observed Ca 2+ spikes are temporally low-pass filtered from the initial depolarization and provide limited information regarding hyperpolarizations and subthreshold events. Direct measurement of transmembrane potential with fluorescent indicators would provide a more accurate account of the timing and location of neuronal activity. Despite the promise of fluorescent voltage-sensitive dyes (VSDs), previous classes of VSDs have each been hampered by some combination of insensitivity, slow kinetics (14-16), heavy capacitative loading (17-21), lack of genetic targetability, or phototoxicity. Two of the more widely used classes of VSDs, electrochromic and FRET dyes, illustrate the problems associated with developing fast and sensitive fluorescent VSDs.Electrochromic dyes respond to voltage through a direct interaction between the chromophore and the electric field (Scheme 1A). This Stark effect leads to small wavelength shifts in the absorption and emission spectrum. Because the electric field directly modulates the energy levels of the chromophore, the kinetics of voltage sensing occur on a timescale commensurate with absorption and emission, resulting in ultrafast (fs to ps) hypso-or bathochromic shifts many orders-of-magnitude faster than required to resolve fast spiking events and action potentials in neurons. This small wavelength shift dictates that the fluorescence signal ...

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“…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). However, combinations of poor sensitivity, slow kinetics, ineffective membrane localization, rapid photobleaching, and/or limited two-photon crosssection, which is important for imaging in thick tissue, have hampered rapid progress toward a general solution for optical voltage imaging.…”

mentioning

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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Anellated Hemicyanine Dyes with Large Symmetrical Solvatochromism of Absorption and Fluorescence

Cited by 56 publications

References 37 publications

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures

Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires

Voltage-sensitive rhodol with enhanced two-photon brightness

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