Fluorescence lifetime predicts performance of voltage sensitive fluorophores in cardiomyocytes and neurons

Voltage imaging with fluorescent indicators offers a powerful complement to traditional electrode or Ca2+-imaging approaches for monitoring electrical activity. Small molecule fluorescent indicators present the unique opportunity for exquisite control over molecular structure, enabling detailed investigations of structure/function relationships. In this paper, we tune the conjugation between aniline donors and aromatic pi systems within the context of photoinduced electron transfer (PeT) based voltage indicators. We describe the design and synthesis of four new voltage-sensitive fluorophores (VoltageFluors, or VFs). Three of these dyes have higher relative voltage sensitivities (Delta F/F) than the previously-reported indicator, VF2.1.Cl. We pair these new indicators with existing VFs to construct a library of voltage indicators with varying degrees of conjugation between the aniline nitrogen lone pair and the aromatic pi system. Using a combination of steady-state and time-resolved fluorescence spectroscopy, cellular electrophysiology, fluorescence lifetime imaging microscopy (FLIM), and functional imaging in mammalian neurons and human cardiomyocytes, we establish a detailed link between the photophysical properties of VF dyes and their ability to report on membrane potential dynamics with high signal-to-noise. Anilines with intermediate degrees of conjugation to the aromatic pi system experience intermediate rates of PeT and possess the highest absolute voltage sensitivities. Measured using FLIM in patch-clamped HEK cells, we find that the absolute voltage sensitivity of fluorescence lifetime (Delta tau(fl) per mV), coupled with traditional fluorescence intensity-based metrics like Delta F/F and signal-to-noise ratio (SNR), provides a powerful method to both predict and understand indicator performance in cellular systems.