• anomalous motion
• Brownian motion
• crowded environment
• FRAP
• microscopy fluorescence techniques
• SPT

Cells control biochemical reactions through two types of compartments: classical and membranelles structures that can concentrate molecules. Since condensate’s formation depends on a delicate balance, disruptions in its regulation can lead to dysfunctional protein assemblies, which may be involved in pathological processes. Therefore, studying biomolecular condensates is fundamental.

Among the available methods for studying droplet’s features, this thesis focuses on two advanced fluorescence microscopy techniques. SPT consists in tracking fluorescent nanometric objects and reconstructing their trajectories at the nanoscale level. The method was validated testing model samples and verifying the Stokes-Einstein law applying it to solutions with increasing concentrations of polyols, in which diffusion is expected to be Brownian. On the other hand, FRAP extracts information from the recovery curve of fluorescence intensity within a region of interest where molecules are photobleached. In this study, we choose a water-glucose solution as a model system to test the technique’s capabilities.

A further step is the investigation of motion inside a crowded environment where it is expected to be anomalous. Using as test systems aqueous solutions of entangled polysaccharides I observed a transition from anomalous to normal diffusion. Moreover, in this case, I found a distribution of displacements that is far from being Gaussian.

Towards the application to biosystems, I demonstrate that the implemented techniques can study processes within biological condensates. An attempt was made, as a proof of principle, in this thesis to track fluorescent objects inside coacervates composed of lysozyme, a digestive enzyme.