Voltage sensing by fluorescence resonance energy transfer in single cells

Abstract

A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C6-(3) and DiSBA-C10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and acceptors from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of approximately 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.

Voltage-sensitive rhodol with enhanced two-photon brightness

“…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.…”

pH-Insensitive FRET Voltage Dyes

“…This experiment was performed using PTS 12 , PTS 14 , and PTS 16 as donors with similar results. PTS 10 was not included due to the destaining phenomenon. Donor and acceptor signals were normalized to the plate background, which in our experience provides a convenient and reproducible fluorescence standard.…”

Cooperative amplification of energy transfer in plasmonic systems

“…Förster resonance energy transfer (FRET) [1,2] between spacially separated donor and acceptor fluorophores, e.g., dye molecules or semiconductor quantum dots (QD), underpins diverse phenomena in biology, chemistry and physics such as photosynthesis, exciton transfer in molecular aggregates, interaction between proteins [3, 4] or, more recently, energy transfer between QDs and in QD-protein assemblies [5][6][7]. FRET spectroscopy is widely used, e.g., in studies of protein folding [8,9], live cell protein localization [10,11], biosensing [12,13], and light harvesting [14].During past decade, significant advances were made in ET enhancement and control by placing molecules or QDs in microcavities [15][16][17] or near plasmonic materials such as metal films and nanoparticles (NPs) [18][19][20][21][22][23][24][25][26][27][28][29][30][31]. While Förster transfer is efficient only for relatively short donor-acceptor separations ∼10 nm [3], a plasmonmediated transfer channel supported by metal NPs [32][33][34][35][36][37], films and waveguides [35,38] or doped monolayer graphene [39], can significant increase the transition rate at larger distances between donor and acceptor.…”

“…Förster resonance energy transfer (FRET) [1,2] between spacially separated donor and acceptor fluorophores, e.g., dye molecules or semiconductor quantum dots (QD), underpins diverse phenomena in biology, chemistry and physics such as photosynthesis, exciton transfer in molecular aggregates, interaction between proteins [3,4] or, more recently, energy transfer between QDs and in QD-protein assemblies [5][6][7]. FRET spectroscopy is widely used, e.g., in studies of protein folding [8,9], live cell protein localization [10,11], biosensing [12,13], and light harvesting [14].…”