A red-emitting carborhodamine for monitoring and measuring membrane potential

Abstract

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes, but also measure values of membrane potentials. This study discloses a new fluorescent indicator which can address both. We describe the synthesis of a new sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials with greater signal-to-noise than state-of-the-art BeRST (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of action potentials in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages. Significance StatementBiological membrane potentials are maintained by all forms of life. In electrically excitable cells, fast changes in membrane potential drive downstream events: neurotransmitter release, contraction, or insulin secretion. The ability to monitor changes in and measure values of cellular membrane potentials is central to a mechanistic understanding of cellular physiology and disease. Traditional modes for measuring membrane potential use electrodes, which are invasive, destructive, low throughput, and ill-suited to interrogate spatial dynamics of membrane potentials. Optical methods to visualize potentials with fluorescent dyes offer a powerful complement to traditional electrode approaches. In this study, we show that a new, red to farred fluorophore can both monitor changes in and measure values of membrane potential in a variety of living systems.