Application of the SPLIT-FLCS Method to the Detection of Nanoscale Diffusion in 3D in Live Cells

Abstract

The diffusion of molecules within the cellular environment has a key role in the spatio-temporal regulation of many cellular processes. The intracellular environment is a complex, heterogeneous and dynamic assembly of macromolecules forming transient structures that can range from several microns down to few tens of nanometers. The presence of these multi-scale structures suggests that diffusion modes might be very different at different spatial scales [1] and that, especially below the diffraction limit, heterogeneities might be related to functional organization. In this respect, optical super-resolution has been combined efficiently with Fluorescence Correlation Spectroscopy (FCS) for investigating molecular diffusion in 2D systems, such as the cellular plasma membrane [2], whereas, unfortunately, its potential has not been fully exploited for investigations in 3D environments.We have recently introduced a low illumination intensity method for super-resolution imaging, based on the separation of fluorophore dynamics rather than fluorescence inhibition [3]. Here we combine this method, named separation of photons by lifetime tuning (SPLIT), with fluorescence lifetime correlation spectroscopy (FLCS) [4], to measure the 3D diffusion of fluorescent proteins at spatial scales tunable from the diffraction size down to 100 nm range in live cells. We discuss the obtained results and the limitations of the method imposed, in live cells, by the simultaneous requirements of minimal photodamage and high signal-to-noise ratio.