Comprehensive fluorescence lifetime fluctuation spectroscopy to investigate protein mobility during aggregation processes in living cells

How proteins move and interact in the complicated, crowded cellular environment is still unknown. The formation of membrane-less condensates, caused by interacting proteins and RNA molecules, in the so-called liquid-to-liquid phase transition has recently raised scientific interest. Primarily related to protein mutations, the condensates can undergo a liquid-to-solid transition often associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Here, we introduce a novel method based on fluorescence fluctuation spectroscopy (FFS) as a tool to study proteins interactions, movement, and aggregate formation comprehensively. FFS is a family of analytical methods that allow quantifying molecular processes by studying the fluorescence intensity fluctuations generated by a population of fluorescent molecules entering and leaving a confined microscope focal volume. We enhance conventional FFS methods by employing a novel single-photon-avalanche-diode array detector in an ISM confocal microscope, which allows us to spatially and temporally tag single photons. Within the same system, we can systematically, and with high throughput, access a wide range of spatial and temporal scales, leading to a comprehensive knowledge of the process. In particular, we can follow slow changes in the overall cell organization by performing time-lapse super-resolved image scanning microscopy, fast-slow molecular movements on the level of single proteins, e.g., time-resolved FFS, and interaction with dual-color cross-correlation methods. Moreover, we introduce fluorescence lifetime fluctuation spectroscopy, a novel paradigm where the molecule's mobility and diffusion mode are simultaneously investigated with fluorescence lifetime, allowing us to directly correlate changes in diffusion mode/mobility with structural changes in the molecule's environment. We validated our methods by measuring the time evolution of stress granules formation, focusing on G3BP1 and FUS proteins, in an in vitro cellular system that recapitulates several features of the ALS pathology.