Resolving Bioactive Supramolecular Structure Formation in Live Cells by Phasor-Fluorescence Lifetime Imaging

Supramolecular polymerization creates transient and life-defining functions in complex cellular systems but synthetically driven analogues has been held back by the lack of real-time correlation. Most synthetically assembled nanostructures in cells are studied when structure formation has reached equilibrium, creating a knowledge divide to the events initiated during the dynamic polymerization phase. Fourier transformed fluorescence lifetime fluctuations allow us to observe these molecular events on the fly, enabling the visualization of the monomers, intermediates, nanostructures and secondary cellular interactions on a 2-D phasor plot. We study the time-lapsed polymerization of amyloid-like naphthalene diimide peptides in MDA-MB 231 cells and show that the growth phase shuts ATP production. The cells then react to overcome the initial stress by mounting an increased respiratory capacity as structure formation nears equilibrium. By coupling simple and fit-free fluorescence lifetime responses to supramolecular chemistry, we reliably detail assembly dynamics and their biological effects with spatiotemporal resolution.