Real-time 4D molecular analysis in dynamic subcellular nanostructures


In many naturally occurring circumstances single-molecules are exerting their functions as part of a nanoscopic system that is continuously and rapidly changing position and shape in time and space. No method exists today that has the capability to fruitfully investigate single-molecule behavior in these conditions, in a 3D environment. The CNI@NEST aims at addressing this challenging task by the development of new fluorescence-based imaging and analysis methods with high spatiotemporal resolution. As strategic platform, we propose to focus an excitation beam in a periodic orbit around the (fluorescently-labeled) nanostructure of interest. The resulting signal will be used as feedback to localize the nanostructure position with an unprecedented combination of spatial (~10 nm) and temporal (~1000 Hz frequency response) resolution. Concomitantly, we are developing novel analytical tools based on spatiotemporal fluorescence correlation spectroscopy (FCS) that provide conceptually the same physical quantities of classical single particle tracking (SPT) techniques but using small, even dim, molecular labels (i.e. with minimal perturbation of the sample), with no need for interpretative models. Of particular note, the ability of these approaches to resolve average molecular dynamic properties well below the limit imposed by diffraction. By adapting and combining this toolbox of analytical approaches along the orbit, single-molecule investigations will become accessible on a moving reference system, for the first time. We believe that this strategy has the potential to develop a flexible, nanotechnology-based, multi-functional approach capable of addressing any high-impact biological question that involves single-molecule behavior on dynamic nanostructures, thus pushing ahead the frontier of current knowledge of living matter.

Pair-correlation methodMolecular Trafficking

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