Fluorescence lifetime predicts performance of voltage sensitive fluorophores in cardiomyocytes and neurons,RSC Chemical Biology

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Fluorescence lifetime predicts performance of voltage sensitive fluorophores in cardiomyocytes and neurons
RSC Chemical Biology ( IF 4.2 ) Pub Date : 2020-12-11 , DOI: 10.1039/d0cb00152j
Steven C Boggess ₁ , Julia R Lazzari-Dean ₁ , Benjamin K Raliski ₁ , Dong Min Mun ₁ , Amy Y Li ₁ , Joshua L Turnbull ₁ , Evan W Miller _{1,

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Affiliation

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 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.

中文翻译：

荧光寿命预测心肌细胞和神经元中电压敏感荧光团的性能

使用荧光指示剂的电压成像为用于监测电活动的传统电极或 Ca ²⁺成像方法提供了强有力的补充。小分子荧光指示剂为精细控制分子结构提供了独特的机会，可以详细研究结构/功能关系。在本文中，我们在基于光致电子转移 (PeT) 的电压指示器的背景下调整苯胺供体和芳香族 π 系统之间的共轭。我们描述了四种新的电压敏感荧光团（VoltageFluors 或 VF）的设计和合成。其中三种染料具有更高的相对电压灵敏度 (Δ F / F) 比之前报告的指标 VF2.1.Cl。我们将这些新指标与现有的 VF 配对，以构建一个电压指标库，在苯胺氮孤对和芳族 π 系统之间具有不同程度的共轭。结合稳态和时间分辨荧光光谱、细胞电生理学、荧光寿命成像显微镜 (FLIM) 以及哺乳动物神经元和人类心肌细胞的功能成像，我们在 VF 染料的光物理特性与其能力之间建立了详细的联系以高信噪比报告膜电位动力学。与芳族 π 系统具有中等共轭度的苯胺经历中等速率的 PeT 并具有最高的绝对电压灵敏度。在膜片钳 HEK 细胞中使用 FLIM 测量，τ _fl per mV)，再加上传统的基于荧光强度的指标，如 Δ F / F和信噪比 (SNR)，提供了一种强大的方法来预测和了解细胞系统中的指标性能。

更新日期：2020-12-12

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