Voltage Imaging with a NIR-Absorbing Phosphine Oxide Rhodamine Voltage Reporter

Abstract

Near infrared (NIR) fluorophores may hold the key for non-invasive optical imaging of deep structures in intact organisms with high spatial and temporal resolution. Yet, developing fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge. Here, we report the design, synthesis, and application of NIR-absorbing and -emitting, sulfonated, phosphine-oxide (po) rhodamines for voltage imaging in thick tissue from the central nervous system. We find po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show best-in class voltage sensitivity (up to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca 2+ -sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca 2+ imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In ex vivo retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca 2+ imaging and traditional multi-electrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

Xanthene dyes for cancer imaging and treatment: A material odyssey

“…In 2021 Gonzalez et al utilized the phospha-rhodamine derivatives for voltage imaging. [86] Four tetraethyl phospha-rhodamine derivatives with methylphosphine oxide as the bridging moiety were synthesized (EtPOMeR-metaX1SO3 -, EtPOMeR-metaX 2 SO 3 -, EtPOMeR-paraX 1 SO 3and EtPOMeR-paraX 2 SO 3 -) to serve as phosphine oxide rhodamine voltage reporters (poRhoVRs, Table 6:E50-55, Scheme 10). All derivatives displayed excitation and emission profiles above 700 nm, were localized to the membrane and displayed differing cellular brightness in HEK cells with good voltage sensitivity.…”

A long-wavelength xanthene dye for photoacoustic imaging

“…This 10' atomic substitution method has been used to design bright, long wavelength fluorescent dyes for various purposes like protein labeling, [12][13] sensing, [14][15] and functional imaging. [16][17][18] Xanthene dyes have been previously explored for PA applications. 4,[19][20][21] The strategies generally involved forcing the excited state dye to decay non-radiatively through the twisted intramolecular charge transfer (TICT) pathway.…”

Chemical Targeting of Rhodol Voltage Sensitive Dyes to Dopaminergic Neurons

“…[25][26][27][28] Synthetic voltage sensors based on various mechanisms have been developed, 26 some examples of which include electrochromic, [29][30][31] semiconductor nanoparticle-based, 32,33 redistribution-based 34 or photoinduced electron transfer (PeT)-based sensors. [35][36][37][38][39][40][41][42][43][44][45] A major shortcoming of synthetic VSDs has been their lack of selectivity for specific neuronal subpopulations. This challenge has been partially tackled by genetically-encoded voltage indicators (GEVIs) -protein-based sensors which optically respond to changes in membrane potential and can be expressed in defined cells via cell-type specific promoters.…”