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.

Chemical Targeting of Rhodol Voltage-Sensitive Dyes to Dopaminergic Neurons

“…A great deal of effort has also been invested in direct optical readout of membrane potential changes with voltage-sensitive dyes (VSDs). − Synthetic voltage sensors based on various mechanisms have been developed, some examples of which include electrochromic, − semiconductor nanoparticle-based, , redistribution-based, or photoinduced electron transfer (PeT)-based sensors. − 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. , Small-molecule dyes, however, have several advantages over protein-based probes, including the availability of a broad palette of structural features for tuning the photophysical properties of the probe. , Hybrid chemo-genetic approaches have successfully fused the advantages of synthetic dyes with those of genetic targeting, either by conjugating voltage-sensitive domains with small-molecule fluorophores − or by enzymatically decaging − or anchoring − synthetic VSDs.…”

Sulfone Rhodamines for Voltage Imaging

“…[5][6][7][8][9][10][11] However, our previous VF dyes have primarily absorbed and emitted within the visible region of light. [5][6][7][8][9][10]12,13] Highly redshifted VF dyes will be essential for the application of these probes to more complex biological systems as lower energy light can penetrate more deeply into biological tissue and reduce background from autofluorescence. [14] Therefore, synthesizing VF dyes with absorbance and emission maxima in the near-infrared region has been a long-standing goal.…”

Late-stage functionalisation of alkyne-modified phospha-xanthene dyes: lysosomal imaging using an off–on–off type of pH probe

“…The phospha-xanthene dyes have been used as NIR-labelling reagents in optical imaging both in vitro and in vivo. 22,27,29 Moreover, following molecular design strategies that have been established for classical xanthene dyes, several phosphaxanthene-based NIR fluorescent probes for the detection of H 2 O 2 , 30 Cu + , 31 Ca 2+ , 32,33 pH changes, 34 and membrane potentials 35 have been reported. In this context, we have successfully developed a series of >P(=O)Ph-substituted dyes, POXs (Fig.…”