Palette of fluorinated voltage-sensitive hemicyanine dyes

Abstract

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440-670 nm; the twophoton excitation range is 900-1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.fluorescence | microscopy O ptical recording techniques provide powerful tools for neurobiologists (1) and cardiac physiologists (2) to study detailed patterns of electrical activity over time and space in cells, tissues, and organs. Rational design methods, based on molecular orbital calculations of the dye chromophores and characterization of their binding and orientations in membranes (3-5), were used to engineer dye structures. The general class of dye chromophores called hemicyanine (also referred to as styryl dyes) has emerged from this effort as a good foundation for voltage-sensitive dyes (VSDs), because they exhibit electrochromism. This mechanism, also referred to as the molecular Stark effect, involves the differential interaction of the electric field in the membrane with the ground and excited states of the dye chromophore. Several important hemicyanine dyes were produced over the years, including di-4-ANEPPS (6, 7), di-8-ANEPPS (8), di-2-ANEPEQ (also known as JPW-1114) (9, 10), RH-421 and RH-795 (11), ANNINE-6 and ANNINE-6+ (12, 13), di-3-ANEPPDHQ (14, 15), di-4-ANBDQBS, and di-4-ANBDQPQ (16,17). Because the electrochromic mechanism is a direct interaction of the electric field with the chromophore and does not require any movement of the dye molecule, all of these dyes provide rapid absorbance and fluorescence responses to membrane potential (V m ); they are, therefore, capable of recording action potentials (APs). Other mechanisms can give more sensitive voltage responses in specialized applications (18)(19)(20)(21)(22). Addit...

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

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“…cades resulted in dyes that report membrane voltage transients with sensitivities exceeding 10 Ϫ1 /100 mV [expressed as change of light intensity (⌬F) over baseline (F), hence ⌬F/F in % for 100-mV change in membrane potential], good photostability, and minimized side effects (Woodford et al 2015;Yan et al 2012). The finding that two-photon (2p) excitation is compatible with low-molecular-weight voltage-sensitive dyes (Fisher et al 2005(Fisher et al , 2008Kuhn et al 2008) opened exciting prospects of voltage imaging from deep brain structures and small neuronal compartments such as dendritic spines (see below).…”