Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein "diffusion law" can be obtained directly from imaging, in the form of a mean-square displacement vs. time-delay plot, with no need for interpretative models. This approach is tested with several simulated 2D diffusion conditions and in live Chinese hamster ovary cells with a GFP-tagged transmembrane transferrin receptor, a well-known benchmark of membrane-skeleton-dependent transiently confined diffusion. This approach does not require extraction of the individual trajectories and can be used also with dim and dense molecules. We argue that it represents a powerful tool for the determination of kinetic and thermodynamic parameters over very wide spatial and temporal scales.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn't need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on µm-sized membrane regions in the micro-to-milli-second time range.

The enormous wealth of information available today from optical microscopy measurements on living samples is often underexploited. We argue that spatiotemporal analysis of fluorescence fluctuations using multiple detection channels can enhance the performance of current nanoscopy methods and provide further insight into dynamic molecular processes of high biological relevance.

The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.

In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale.