Optical counting in live cell nanoscopy

The spatial organisation of signalling molecules in macromolecular assemblies is an important regulator of cellular processes.

The transient nature and variable composition of them demands imaging methods able to read protein copy number while following their dynamics.

Super resolution fluorescence microscopies are essential to unveil their nanoscale organization in the crowded cellular environment.

However, the quantification of number of molecules across time remain difficult to achieve due to photo-bleaching and slow recording time, with often only one single time point accessible.

My aim is to develop a molecular counting method compatible with live cell imaging (~1 Hz speed) and able to reach nanoscale resolution (~50 nm). The method will be based on the reversible switching ability of smart fluorescent proteins.

The photo-switching introduces an additional statistical dependency in the image formation that will allow to translate intensity into number of emitters. Furthermore, it is compatible with RESOLFT nanoscopy, suitable for 3D and time lapse imaging of living cells.

The project will include the development of a new theoretical framework for molecular counting together with its experimental demonstration on nanotemplates and in cells, with specifically designed labelling strategies.

Finally, the method will be employed to investigate how the dynamics of the molecular and structural complexity of receptors in immune cells underlies their activation.