Intracellular Protein Scaffolds Enable Simultaneous Measurement of Multiple Biological Signals From Spectrally Identical Fluorescent Sensors

Abstract

Biological signals interact in complex ways within cells, and can exhibit great cell-to-cell heterogeneity as a function of cell history and state. Therefore, there is increasing desire to use multiple fluorescent sensors to simultaneously image multiple biological signals at the same time in individual cells. For decades the limited number of sensors recorded simultaneously has been due to spectral overlap. To circumvent this limitation of spectrally multiplexing sensors, molecular tools for spatially multiplexing have been engineered. Three biological signals were simultaneously measured with spectrally-overlapping sensors using these novel molecular tools. This initial demonstration of the spatial multiplexing strategy opens the door for the simultaneous imaging of dozens of signals within a physiological cascade as more peptide sequences for clustering are designed.