Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging

Kanuj Mishra,Andriy Chmyrov,Vasilis Ntziachristos,Juan Pablo Fuenzalida-Werner,Robert Janowski,Ilaria Testa,Christian Zakian,Uwe Klemm,Andre C Stiel,Dierk Niessing,Francesca Pennacchietti,Yuanhui Huang

doi:10.1038/s41587-021-01100-5

Abstract

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

Highlights

Switchable fluorescent proteins (RSFPs) can be alternated between a fluorescent and nonfluorescent state by illumination with light of varying wavelength
This behavior is similar to green Reversibly switchable fluorescent proteins (RSFPs) such as Dronpas and rsEGFPs (Fig. 1f), with the 488-nm band readily photo-switchable
Excited-state lifetime measurements and global biexponential fitting of fluorescent switching at different Ca2+ concentrations suggest that at least two distinct species are involved in tailoring the Ca2+ response (Supplementary Fig. 6, Supporting Information 2 and Extended Data Figs. 1 and 2)

Summary

Introduction

Switchable fluorescent proteins (RSFPs) can be alternated between a fluorescent and nonfluorescent state by illumination with light of varying wavelength. GEIs are proteins in which the signal from a readout moiety is altered following binding of a receptor moiety to a target chemical This mechanism allows for the use of GEIs in visualization of the dynamic chemical composition of cells and their surroundings, enabling sensing of small molecules, including metabolites and ions[15] such as Ca2+ Augmention of GEIs with photo-switching capabilities (rsGEIs) would enable both the visualization of chemical distributions at the nanoscale using RESOLFT SRM concepts, as well as the sensing of molecules and ions in OA imaging of whole live animals, by overcoming tissue background absorption. These sensors show photo-switching only when bound to the molecule of interest

Methods

Results

Conclusion