Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS

Abstract

The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details of the subcellular topology and its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale diffusion (STED-FCS). However, stimulated-emission depletion fluorescence correlation spectroscopy has only been used successfully to reveal functional organization in two-dimensional space, such as the plasma membrane, while, an efficient implementation for measurements in three-dimensional space, such as the cellular interior, is still lacking. Here we integrate the STED-FCS method with two analytical approaches, the recent separation of photons by lifetime tuning and the fluorescence lifetime correlation spectroscopy, to simultaneously probe diffusion in three dimensions at different sub-diffraction scales. We demonstrate that this method efficiently provides measurement of the diffusion of EGFP at spatial scales tunable from the diffraction size down to ∼80 nm in the cytoplasm of living cells.

Advances in Imaging Plant Cell Dynamics

“…For quantitative approaches such as FRAP, FRET, FLIM, and FCS, the CLSM apparatus remains the workhorse, although other methods may be used instead (i.e. SIM can be combined with FRAP [Conduit et al, 2015] and STED can be used for FCS [Lanzanò et al, 2017]). With no doubt, recently developed advanced superresolution methods (such as SIM, PALM, STORM, and STED, software-based superresolution approaches, and Airyscan CLSM) and methods for long-term developmental imaging (such as LSFM and SPIM) have provided new dimensions and perspectives for better imaging of plant cells, especially in terms of significantly improved spatial and temporal resolution.…”

“…5, F-I). A positive feature of STED is that, being a confocal microscopy-based technology, it can be used for superresolution in combination with quantitative imaging methods such as fluorescence correlation spectroscopy (FCS) either at the cell surface (Andrade et al, 2015) or in deeper parts of the cell (Lanzanò et al, 2017). Unlike SIM and PALM/STORM, which require postacquisition image processing to deliver the superresolved result, STED provides superresolution during acquisition.…”

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

“…Even if limited by diffraction, these methods detect differences in the diffusion of nanometer‐sized probes and can be used to indirectly infer properties of the nanoscale chromatin architecture . FCS can be eventually coupled with STED to probe diffusion at subdiffraction spatial scales . Other examples are fluorescence lifetime imaging (FLIM) microscopy and fluorescence anisotropy imaging, which have been used to monitor alterations in the local environment of a fluorescent probe and relate them to the chromatin condensation state .…”

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

“…Fluorescence correlation spectroscopy (FCS) has, since its introduction almost 50 years ago, become a widely applied technique to study diffusion dynamics in synthetic and biological applications [1][2][3] . It has greatly contributed to the understanding of molecular diffusion in model systems and living cells, both in 2D (in vitro models or cellular membranes) and in 3D (solution or cellular cytoplasm and nucleus) environments [4][5][6][7][8] . Notably, it has offered fundamental insights into the dynamic organisation of living systems at the molecular level, e.g.…”