STARSS: selective time resolved anisotropy with reversible switchable states

Molecular binding, enzymatic activity, and protein complex formation are constantly happening in our cells.

To detect such event we need to precisely and efficiently label the molecule undergoing such activities and possibly resolve them with 1 nm spatial resolution and fast enough to find and follow them over time.

Even with the most advanced super resolution light microscope is hard to study molecular interaction over time due to the insufficient spatial and temproal resolution.

FRET is routinely used to study molecular interaction but when applied to live cells presents several limitation including the low labelling efficiency, noise and poor FRET efficiency.

FCS can probe molecular interaction but it fails when the complex is too large or in densely packed cellular regions where signal fluctuations are barely detectable. Additionally, it is hard to scale up these methods for high throughput studies such as screening applications. Here.

I aim to fill this methodological gap by developing STARSS, a new approach to measure rotational diffusion of large molecules in cells. This method builds on the theory of time resolved anisotropy (TRFA) adding the fluorescence photo-switching component.

With these new probes, recently developed in my lab, I will extend the temporal window of TRFA recordings to monitor the rotation of large proteins and their complexes in situ. A proof of principle demonstration in cellular organelles and viral like particles will be provided.