Spectral Detection Enables Multi-Color Fluorescence Fluctuation Spectroscopy Studies in Living Cells

Abstract

Fluorescence fluctuation spectroscopy techniques provide a powerful toolbox to quantify transport dynamics and interactions of biomolecules in living cells. Cross-correlation analysis of spectrally well-separated fluctuations allows to investigate hetero-interactions between different biomolecules. This analysis is conventionally limited to two fluorescent species that are excited with one or two laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we implement a methodology to investigate such interactions at the plasma membrane (PM) of living cells, in the context of e.g. viral assembly or receptor-ligand interactions. To this aim, we introduce scanning spectral fluorescence correlation spectroscopy (SSFCS), a combination of scanning FCS with spectrally resolved detection and decomposition, inspired by fluorescence lifetime correlation spectroscopy. We first demonstrate that SSFCS allows cross-talk free cross-correlation analysis on cells expressing spectrally highly overlapping fluorescent proteins (FPs) such as mEGFP and mEYFP at the PM, with a single excitation line. We then verify the performance of SSFCS for the quantification of diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we show the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2 as overlapping FP tags in the red spectral region. Using this set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species, and successfully demonstrate multiplexed RSICS measurements of protein interactions in cellular cytoplasm. Finally, we apply SSFCS and RSICS to investigate the assembly of the Influenza A virus matrix proteins and ternary polymerase complex at the PM and in the nucleus of living cells, respectively.