Spectra, membrane binding, and potentiometric responses of new charge shift probes

Abstract

The properties of a series of new potentiometric membrane probes have been explored. The probes all contain an (aminostyryl)pyridinium chromophore or a more highly conjugated analogue. The spectral properties of the dyes are discussed in terms of the excitation-induced charge shift from the pyridine to the aniline; this charge shift also provides the basis for the voltage dependence of the spectra according to an electrochromic mechanism. The spectral responses to a membrane potential on a hemispherical bilayer have been obtained and, grossly, are quite similar for all probes tested. The more subtle variations from dye to dye can be partially rationalized by consideration of binding parameters, the depth within the membrane, and structural factors. The most potential sensitive dye in this collection has been designated di-4-ANEPPS and has a 6-amino-2-naphthyl group in place of the p-anilino on the parent chromophore. Both the relative fluorescence emission and excitation responses have maxima of 8% per 100 mV, and these two spectra display a striking symmetry.

2‐photon excitation fluorescence microscopy enables deeper high‐resolution imaging of voltage and Ca2+ in intact mice, rat, and rabbit hearts

“…This, coupled with the availability of voltage-and Ca 2+ -sensitive dyes that fluoresce after 2P excitation [13,14] allowed us to develop protocols for high-resolution deep imaging of AP and intracellular Ca 2+ characteristics of in situ cardiomyocytes in intact, perfused whole hearts. These dyes include the aminonaphthylethenylpyridinium (ANEP) dyes, which bind to the plasma membrane of cells and undergo an emission spectral shift upon V m changes [15], and Ca 2+ -sensitive Fura dyes, which change excitation spectral properties relative to intracellular Ca 2+ [16].…”

Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol

“…15 Because of their effectiveness in other dyes, butyl groups were selected to maximize tissue retention without an excessive compromise on water solubility. 14 The depth of chromophore penetration into the lipid bilayer also increases with the number of carbons between it and the covalently linked anchor.…”

“…Most styryl VSDs dyes require an additional reagent such as DMSO, Pluronics or cyclodextrins for transport through the aqueous medium to the tissues. 9,15 Labeling conditions are determined for each dye, which can be a time-consuming, trial and error process. 4 Attachment of an uncharged hydrophilic anchoring group to the dye structure may eliminate the need for such agents.…”

Image Informatics Strategies for Deciphering Neuronal Network Connectivity

“…extracted from mouse embryos), bust, synchronized network activity 14,18 . Neuronal electrical activity can be visualized by means of voltage or calcium sensors, both of which are available as synthetic dyes or genetically encoded fluorescent proteins [30][31][32][33] . Such a functional approach not only allows assessing the effect of chronic treatments on neuronal connectivity, but can also provide information about acute responses to pharmacological perturbations.…”

“…Classical voltage sensors such as potential sensitive aminonaphthylethenylpyridinium (ANEP) dyes display a spectral shift upon a change in voltage across the membrane 31 ; more recently developed genetically encoded sensors, such as FlaSh 89 , ElectricPk 90 or ArcLight 32,91 , change intensity with voltage. Despite rapid developments in the field 92 , voltage sensors still do not cover a very high dynamic range and typically have to be measured very fast (up to 60 kHz).…”