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

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 specimen's 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 comprehensive 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 proposed 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.

ALS-associated FUS mutation reshapes the RNA and protein composition of Stress Granules

“…Fluorescence Correlation Spectroscopy (FCS) measurements were performed as described in (36, 70). Cells were plated and FUS expression was induced as previously described; before starting the acquisition, culture medium was replaced with FluoroBriteTM DMEM (Thermo Fisher, A1896701) supplemented with 10% FBS, and sodium arsenite was added at a final concentration of 0.5 mM for the “stressed” samples.…”

M6A reduction relieves FUS-associated ALS granules

“…To perform the FCS measurements, we used a custom laser-scanning microscope equipped with a 5x5 SPAD array detector described by Perego et al 30 .…”

“…4d). We also employed a variation of conventional fluorescence correlation spectroscopy, utilizing a single-photon avalanche diode (SPAD) array detector 29,30 . This innovative approach enables comprehensive studies of molecular mobility within living cells.…”

“…To perform the FCS measurements, we used a custom laser-scanning microscope equipped with a 5x5 SPAD array detector described by Perego et al 30 . Cells were seeded onto a μ-Slide 8 well plate (Ibidi GmbH) and imaged in Live-Cell Imaging Solution (ThermoFisher Scientific) at 37°C.…”

“…The individual correlation curves were visually inspected, and all curves without artifacts were averaged and fitted with a function describing a 3D diffusion. The diffusion coefficients are calculated from the fitted diffusion times knowing the confocal volumes of the microscope (calibrated as described by Perego et al 30 ). Once the diffusion coefficients were calculated, the confinement strength is simply quantified by dividing the diffusion coefficient of the correlation of the signal from the central pixel only with the one retrieved from the correlation of the Sum 55 pixels.…”