Abstract
Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, thus achieving very good statistics but at only one point in the cell. Earlier image-based methods including raster-scan and spatiotemporal image correlation need spatial averaging over relatively large areas, thus compromising spatial resolution. Here, we use spatial pair-cross-correlation in two dimensions (2D-pCF) to obtain relatively high resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of the average path followed by molecules is created.
Highlights
Signal transduction at a sub-cellular level is a key element in the regulation of cell functions, cell fate and cell migration
We have shown connectivity maps for measurements done for simulated molecules moving in a channel and enhanced green fluorescent protein (EGFP) molecules diffusing in physical grooves where we had a clear expectation of the path followed by the molecules
In this paper we describe a method based on the 2D-pCF to produce high resolution maps of molecular movements in cells
Summary
Signal transduction at a sub-cellular level is a key element in the regulation of cell functions, cell fate and cell migration. 2D-pCF (Eq (1)) detects barriers to diffusion and heterogeneity because the time of the correlation maximum is delayed in the presence of diffusion barriers In a nutshell, this non-invasive, sensitive technique can follow the same molecule over a large area producing a map of molecular diffusion. From the technological perspective we need very high resolution spatial sampling coupled with a large field of view, and very high time resolution coupled with high sensitivity detection in order to measure the fluctuations due to fast moving and dim molecules in a whole cell. This can be achieved using fast and highly sensitive cameras. The practical demonstration of connectivity maps may result in a breakthrough in the study of biochemical communication in live cells, by unveiling the role of the cell spatial organization in signal spreading, as well as describing the route followed by the signal molecules to spread from the source to the target
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