Optically Modulated Fluorescent Proteins Enhance Sensitivity in Live Cell Imaging

Abstract

Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular structure and processes. Although the palette of fluorescent proteins has revolutionized detection and dynamics in molecular and cellular biology, the limited brightness, low spectral discrimination, and high cellular autofluorescence continue to limit applications. In contrast to photoswitch-based methods, we have developed a spectroscopic method to recover fluorescence from photoaccessible dark states via long-wavelength secondary laser co-illumination. We selectively recover the higher energy fluorescence by modulating this secondary laser without modulating background autofluorescence. Using hypothesized photoreversible isomerizations of the chromophore, we have identified specific fluorescent protein mutants, ranging from blue to red, capable of optical modulation. Employing these methods, we have demonstrated the ability to recover the modulated signal, with >10-fold improvement, from background in live cells. Such modulation schemes enable new imaging modalities for probing intracellular kinetics and equilibria of low-abundance proteins.