Tracking of individual freely diffusing fluorescent protein molecules in the bacterial cytoplasm

TL;DR

This paper presents a method combining super-resolution fluorescence imaging and stochastic photoactivation to track individual protein molecules in bacterial cells with high temporal and spatial resolution.

Contribution

It introduces a novel imaging approach enabling detailed tracking of single proteins in live bacteria, facilitating studies of intracellular dynamics.

Findings

mEos2 diffuses normally in bacterial cytoplasm

Diffusion coefficient of mEos2 is approximately 13.1 μm^2/s

Method allows for studying single-molecule binding events

Abstract

We combine stroboscopic laser excitation with stochastic photoactivation and super-resolution fluorescence imaging. This makes it possible to record hundreds of diffusion trajectories of small protein molecules in single bacterial cells with millisecond time resolution and sub-diffraction limited spatial precision. We conclude that the small protein mEos2 exhibits normal diffusion in the bacterial cytoplasm with a diffusion coefficient of 13.1 -+ 1.2 \mu m^2 s^(-1). This investigation lays the groundwork for studying single-molecule binding and dissociation events for a wide range of intracellular processes.