Mini-Symposium: "Array detectors, the future of confocal microscopy is now!"

Summary: Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we present a photon-resolved microscope to provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. The platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse imaging with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales.

Short bio: Eleonora Perego studied Physics at the University of Milano-Bicocca (Italy) where she got a M. Sc. in Physics in 2015. Then she moved to the University of Goettingen where she obtained in 2020 a PhD in Biophysics, titled “Studying molecular interaction under flow with fluorescence fluctuation spectroscopy”, working in the group of Sarah Koester. Since 2021, Eleonora was at the Italian Institute of Technology in Genoa (Italy) in the Molecular Microscopy and Spectroscopy research line of Giuseppe Vicidomini. Her research interests include the application of fluorescence spectroscopy methods, such as fluorescence fluctuation spectroscopy and fluorescence lifetime imaging, to biological questions, ranging from neuroscience to RNA biology. Now, she just started a position where she will be using the microscopy methods develops so far understanding the role of condensation during transcription activation.

Summary:Single-molecule localization microscopy using MINFLUX enables nanometer precision localization of individual molecules with a minimal number of detected photons. However, its implementation requires a complex and costly setup. We introduce ISM-FLUX, a novel localization strategy that integrates the MINFLUX concept with a small array detector, allowing it to be implemented on a conventional confocal microscope with minimal modifications. Our proof-of-concept demonstrations on various samples consistently achieve resolutions below 20 nm. In summary, ISM-FLUX provides a user-friendly and less complex alternative to MINFLUX, delivering comparable performance.

Short bio: Eli Slenders earned his PhD from Hasselt University with a thesis entitled "Resolution in Coherent and Incoherent Optical Imaging with Two–Photon Excitation Microscopy," under the supervision of Prof. Dr. M. Ameloot. In 2019, he joined the Molecular Microscopy and Spectroscopy lab at the Italian Institute of Technology in Genoa as a postdoctoral researcher, working with Giuseppe Vicidomini. He currently continues his research in the same group, focusing on the development of novel optical microscopy techniques for single-molecule localization.

Abstract: Biomolecular condensation via liquid-liquid phase separation (LLPS) is crucial for orchestrating cellular activities temporospatially. Although the rheological heterogeneity of biocondensates and the structural dynamics of their constituents carry critical functional information, methods to quantitatively study biocondensates are lacking. Single-molecule fluorescence research can offer insights into biocondensation mechanisms. Unfortunately, as dense condensates tend to sink inside their dilute aqueous surroundings, studying their properties via methods relying on Brownian diffusion may fail. We take a first step toward single-molecule research on condensates of Tau protein under flow in a microfluidic channel of an in-house developed microfluidic chip. Fluorescence correlation spectroscopy (FCS), a well-known technique to collect molecular characteristics within a sample, was employed with a newly commercialized technology, where FCS is performed on an array detector (AD-FCS), providing detailed diffusion and flow information.

Short bio: Stijn Dilissen is a PhD student in the Dynamic Bioimaging Lab under the supervision of Professor Jelle Hendrix. He is an interdisciplinary biomedical researcher focusing on nanotechnology and advanced light microscopy. Stijn specializes in developing microfluidic approaches for biomolecular condensate research and artificial cell devices to study drug-target interactions, particularly protein dynamics, using smFRET microscopy. He holds a Master's degree in Biomedical Sciences, specializing in Bioelectronics and Nanotechnology.