Highly multiplexed imaging of biosensors in live cells

Abstract

Fluorescent biosensors allow for real-time monitoring of biochemical activities in cells, but their multiplexing capacity is severely limited by the availability of spectral space. We overcome this problem by developing a set of barcoding proteins that are spectrally separable from commonly used FRET (fluorescence resonance energy transfer)-based and single-fluorophore biosensors. Mixed populations of barcoded cells expressing different biosensors can be concurrently imaged and computationally unmixed to achieve highly multiplexed tracking of biochemical activities in live cells.

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

“…Multiplex live-cell imaging approaches are agile techniques that allow simultaneous multicolor imaging experiments in live cells co-expressing different biosensors [ 14 , 16 ]. In a recent study, researchers developed a multiplex imaging approach employing post hoc spectral unmixing analysis by exploiting targeted blue and red fluorescent proteins as a barcoding system that permits recording up to 72 combinations of fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors [ 17 ]. Another approach uses single-color probes for obsolete complex and laborious spectral unmixing and image processing.…”