Spatial triple-correlation spectroscopy reveals heterotrimer dynamics in live cells

Abstract

Heterotrimeric protein complexes are central regulators of intracellular signaling, yet their dynamic assembly and transport in living cells remain difficult to resolve. Here, we present spatial triple-correlation spectroscopy (S3CS), a fluorescence fluctuation method that integrates three-channel line scan microscopy with a spatial triple-correlation function to directly detect fluorescent heterotrimers and map their movement relative to subcellular architecture. Simulations establish that S3CS quantitatively captures heterotrimer formation, local diffusion, and long-range transport, while live-cell experiments confirm its specificity for following fluorescent ternary assemblies in the pres..