Genetically encoded fluorescent biosensors convert specific biomolecular events into optically detectable signals. However, imaging biomolecular processes often requires modifying the proteins involved, and many molecular processes are still to be imaged. Here, we present a biosensor design that uses a hitherto overlooked detection principle: directionality of optical properties of fluorescent proteins. The biosensors (termed FLIPs) offer an extremely simple design, high sensitivity, multiplexing capability, ratiometric readout, and other advantages, without requiring modifications to their targets. We demonstrate the sensor performance by real-time imaging activity of G protein-coupled receptors (GPCRs), G proteins, arrestins, and other membrane-associated proteins, as well as by identifying a previously undescribed, pronounced, endocytosis-associated conformational change in a GPCR-β-arrestin complex. In combination with an original tri-scanning linear dichroism confocal microscope, FLIPs allow unparalleled imaging of activity of nonmodified, endogenously expressed G proteins. Thus, FLIPs establish a powerful molecular platform for imaging cell signaling, allowing numerous future developments and insights.

1st Faculty of Medicine Charles University Prague Kateřinská 32 121 08 Prague Czechia

Department of Biochemistry Molecular and Structural Biology KU Leuven Celestijnenlaan 200G 3001 Heverlee Belgium

Innovative Bioimaging s r o Podolská 1490 6 147 00 Prague 4 Czechia

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nám 2 160 00 Prague Czechia

Institute of Plant Molecular Biology Biology Center of the Czech Academy of Sciences Branišovská 1160 30 Budweis Czechia

Université de Lille LASIRe CNRS 59655 Villeneuve France

University of South Bohemia Faculty of Science Branišovská 1160 30 Budweis Czechia

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