Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons

Abstract

We present a method to target voltage-sensitive fluorescent dyes to specified cells using an enzyme-catalyzed fluorogenic reaction on cell surfaces. The dye/enzyme hybrids are composed of a photoinduced electron transfer (PeT)-based fluorescent voltage indicator and complementary enzyme expressed on the cell surface. Action of the exogenous enzyme on the dye results in fluorogenic activation of the dye, enabling fast voltage imaging in defined neurons, with sensitivity surpassing purely genetically-encoded approaches. We employ a bulky cyclopropylmethyl acetoxymethyl ester to diminish the fluorescence of a PeT-based voltage-sensitive dye, or VoltageFluor. The hydrolytically stable ester can be removed by the action of porcine liver esterase (PLE) to reveal the bright, unmodified VoltageFluor. We established that the chemically modified VoltageFluor is a substrate for PLE in vitro and in live cells. When PLE is targeted to the external face of cell membranes, it controls the apparent staining of cells. Use of neuron-specific promoters can direct staining to mammalian neurons to provide clear detection of neuronal action potentials in single trials. All of the new VoltageFluors targeted by esterase expression report single spikes in cultured mammalian neurons. The best, VF-EX2, does so with a signal-to-noise ratio (SNR) nearly double that of comparable genetically-encoded voltage reporters. By targeting PLE to neurons, VF-EX2 can interrogate the neuromodulatory effects of serotonin in cultured hippocampal neurons. Together, we show that a combination of synthetic chemistry and biochemistry enables bright and fast voltage imaging from genetically-defined neurons in culture.

Recording Neural Activity Based on Surface Plasmon Resonance by Optical Fibers-A Computational Analysis

“…Optical methods of recording neural activity and stimulation of neurons are performed using different techniques. Infrared excitation (Wells et al, 2005 ; Shapiro et al, 2012 ), voltage sensitive fluorescent tags (Kulkarni and Miller, 2017 ; Liu et al, 2017 ; Nixima et al, 2017 ), genetically encoding opsins (Chow et al, 2012 ), and surface plasmon resonance techniques (Kim et al, 2008 ) are from well-known methods in this regard. Fluorescence-based techniques using voltage sensitive dyes suffer from different problems and difficulties including bleaching, toxicity, labeling procedures and the instrumentation requirements.…”

Optical determination of absolute membrane potential

“…In principle, VF-FLIM could record subcellular differences in V mem that would be difficult to dissect with electrophysiology. Looking ahead, such subcellular recordings in cells with complex morphology and processes are an exciting area for future development of VF-FLIM, in conjunction with cellular and sub-cellular strategies for targeting VF dyes 38,39 .…”

“…VF-FLIM represents a novel and general approach for interrogating the roles of membrane potential in fundamental cellular physiology. Future improvements to the voltage resolution could be made by use of more sensitive indicators, which may exhibit larger changes in fluorescence lifetime 24 VF-FLIM can be further expanded to include the entire color palette of PeT-based voltage indicators 57,58 , allied with targeting methods to probe absolute membrane potential in heterogeneous cellular populations 38,39 , and coupled to high-speed imaging techniques for optical quantification of fast voltage events 59 .…”

Making Life Visible: Fluorescent Indicators to Probe Membrane Potential

“…Based on the success of the photoactivation approach for staining single cells, we envisioned a similar strategy could be employed for enzymatic activation of voltagesensitive dye, providing contrast without the need for a separate photoactivation step (Liu et al 2017). To achieve targeting, the parent VF dye is chemically modified to be minimally fluorescent and must be enzymatically activated prior to imaging ( Fig.…”

“…This is due in part to the improved membrane localization of uncaged VF-EX dyes. Whereas a significant fraction of expressed fluorescent GEVIs remains in the cytosol and internal compartments, contributing to unresponsive background fluorescence, the genetically-encoded component PLE is non-fluorescent and the dye only Liu, Grenier, Hong, Muller, andMiller, J. Am.…”