Quantitative evaluation of methods to analyze motion changes in single-particle experiments Muñoz-Gil, G., H. Bachimanchi, J. Pineda, B. Midtvedt, G. Fernández-Fernández, B. Requena, Y. Ahsini, S. Asghar, J. Bae, F. J. Barrantes, S. W. B. Bender, C. Cabriel, J. A. Conejero, M. Escoto, X. Feng, R. Haidari, N. S. Hatzakis, Z. Huang, I. Izeddin, H. Jeong, Y. Jiang, J. Kæstel-Hansen, J. Miné-Hattab, R. Ni, J. Park, X. Qu, L. A. Saavedra, H. Sha, N. Sokolovska, Y. Zhang, G. Volpe, M. Lewenstein, R. Metzler, D. Krapf, G. Volpe, and C. Manzo Nature Communications 16, no. 1 (2025)
Résumé: The analysis of live-cell single-molecule imaging experiments can reveal valuable information about the heterogeneity of transport processes and interactions between cell components. These characteristics are seen as motion changes in the particle trajectories. Despite the existence of multiple approaches to carry out this type of analysis, no objective assessment of these methods has been performed so far. Here, we report the results of a competition to characterize and rank the performance of these methods when analyzing the dynamic behavior of single molecules. To run this competition, we implemented a software library that simulates realistic data corresponding to widespread diffusion and interaction models, both in the form of trajectories and videos obtained in typical experimental conditions. The competition constitutes the first assessment of these methods, providing insights into the current limitations of the field, fostering the development of new approaches, and guiding researchers to identify optimal tools for analyzing their experiments.
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3D Single-Molecule Super-Resolution Imaging of Microfabricated Multiscale Fractal Substrates for Calibration and Cell Imaging Cabriel, C., R. M. Córdova-Castro, E. Berenschot, A. Dávila-Lezama, K. Pondman, S. Le Gac, N. Tas, A. Susarrey-Arce, and I. Izeddin ACS Applied Materials and Interfaces 17, no. 6, 9019-9034 (2025)
Résumé: Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open up new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity as well as axial calibration issues. In this work, we address this issue by leveraging the geometrical characteristics of fractal-like structures, which serve as axial calibration tools and modulate cell growth. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as an alternative to electron microscopy for material imaging but also as calibration tools for 3D super-resolution microscopy. We used their multiscale and known geometry to perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. We then utilized these substrates as a platform for high-resolution bioimaging. As a proof of concept, we cultivate human mesenchymal stem cells on these substrates, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with a 3D SMLM assessment.
Mots-clés: 3D single-molecule localization microscopy; bioimaging; multiscale material; fractal-like microstructures; calibration; material imaging
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