Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Perego, E.; Zappone, S.; Castagnetti, F.; Mariani, D.; Vitiello, E.; Rupert, J.; Zacco, E.; Tartaglia, G. G.; Bozzoni, I.; Slenders, E.; Vicidomini, G.

doi:10.1101/2023.06.09.544221

bioRxiv · 10.1101/2023.06.09.544221

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Perego, E.·Zappone, S.·Castagnetti, F.·Mariani, D.·Vitiello, E.·Rupert, J.·Zacco, E.·Tartaglia, G. G.·Bozzoni, I.·Slenders, E.·Vicidomini, G.

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Abstract

Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimens fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies - compatible with imaging - to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation - inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes - not limited to liquid-liquid phase transition - and we anticipate their widespread application in future life-science research.

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Perego, E., Zappone, S., Castagnetti, F., Mariani, D., Vitiello, E., Rupert, J., Zacco, E., Tartaglia, G. G., Bozzoni, I., Slenders, E., Vicidomini, G.. 2023-06-11. Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation. https://doi.org/10.1101/2023.06.09.544221

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