# Polarized Phase-Sensitive Fluorescence-Image Correlation Spectroscopy

## TL;DR

This paper introduces a new imaging technique that combines fluorescence lifetime and polarization to study molecular interactions in living cells.

## Contribution

The novel contribution is integrating fluorescence lifetime imaging with image correlation and polarization analysis to measure molecular assembly properties in heterogeneous systems.

## Key findings

- The method successfully extracts cluster densities, brightnesses, and rotational correlation times in a model system.
- A two-population model demonstrates the feasibility of the approach in heterogeneous environments.
- The technique provides metrics for concentration, size, and dynamics of molecular assemblies.

## Abstract

Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of these molecular assemblies remains an enduring biophysical challenge, particularly in cells, where heterogeneity is the rule rather than the exception. Orthogonal signals derived from fluorescence lifetime, fluorescence fluctuations, and fluorescence polarization provide valuable metrics for probing interactions and environments, concentration and size, and rotational dynamics, respectively. This paper combines fluorescence lifetime imaging microscopy with image correlation analysis and polarization to determine the concentrations, brightness, lifetime, and rotational correlation time of different fluorescent states. A two-population model is examined as a prototypical example of a heterogeneous system. The analysis is illustrated on a simple fluorescence model system, where cluster densities, relative brightnesses, lifetimes, and rotational correlation times are extracted.