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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 Ca(2+)-imaging approaches for monitoring electrical activity. Small molecule fluorescent indicators present the unique opportunity for exquisite control over molecular structure, enabling detailed in...

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Detalles Bibliográficos
Autores principales: Boggess, Steven C., Lazzari-Dean, Julia R., Raliski, Benjamin K., Mun, Dong Min, Li, Amy Y., Turnbull, Joshua L., Miller, Evan W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: RSC 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240514/
https://www.ncbi.nlm.nih.gov/pubmed/34212146
http://dx.doi.org/10.1039/d0cb00152j
Descripción
Sumario:Voltage imaging with fluorescent indicators offers a powerful complement to traditional electrode or Ca(2+)-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 π 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 (ΔF/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 π 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 π 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 (Δτ(fl) per mV), coupled with traditional fluorescence intensity-based metrics like ΔF/F and signal-to-noise ratio (SNR), provides a powerful method to both predict and understand indicator performance in cellular systems.