Molecular prosthetics for long-term functional imaging with fluorescent reporters

Abstract

Voltage-sensitive fluorescent reporters can reveal fast changes in membrane potential in neurons and cardiomyocytes. However, in many cases, illumination in the presence of the fluorescent reporters results in disruptions to action potential shape that limits the length of recording sessions. We show here that a molecular prosthetic approach, previously limited to fluorophores, rather than indicators, can be used to substantially prolong imaging in neurons and cardiomyocytes. IntroFluorophores are indispensable in the investigation of living systems. The problem is they bleach and can perturb the very system they are meant to observe. Bleaching makes it difficult to make sustained measurements, and phototoxicity introduces artifacts by disrupting the underlying physiology of the biological system. Exogenous photoprotectant cocktails, often containing anti-oxidants or triplet-state quenchers (TSQs), are often added to imaging media to prolong imaging duration by either reducing photobleaching or phototoxicity. Pioneering work showed that intramolecular tethering of a TSQ, like vitamin E derivative Trolox, or cyclooctatetrane (COT), profoundly improves the photostability and reduces phototoxicty of common fluorophores in single molecule 1 and cellular imaging. 2 However, this self-healing fluorophore strategy has never been applied to fluorescent reporters: dyes which change their optical properties in response to biological cues, such as pH, Ca 2+ , or membrane potential. Additionally, because the precise molecular mechanisms behind self-healing fluorophores remain under investigation, 3,4 it was not clear whether the presence of a TSQ would interfere with the sensing mechanism of the fluorescent reporter.