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High‐Speed Interferometric Scattering Tracking Microscopy of Compartmentalized Lipid Diffusion in Living Cells
Tekijät: Reina, Francesco; Eggeling, Christian; Lagerholm, Christoffer
Kustantaja: Wiley
Julkaisuvuosi: 2025
Lehti:: ChemPhysChem
Artikkelin numero: e202400407
ISSN: 1439-4235
eISSN: 1439-7641
DOI: https://doi.org/10.1002/cphc.202400407
Verkko-osoite: https://doi.org/10.1002/cphc.202400407
Rinnakkaistallenteen osoite: https://research.utu.fi/converis/portal/detail/Publication/504617930
Lateral diffusion measurements have been -used to infer information about the nano-organization of membranes. We employed interferometric scattering (ISCAT) microscopy at an acquisition rate of 2 kHz to revisit the diffusion dynamics of a phospholipid analog on the plasma membrane of Ptk2 cells. The ISCAT trajectory data are analyzed with an unbiased, statistics-driven pipeline to identify the most likely diffusion mode from a set of plausible diffusion modes. At the ensemble average level, the data are best described as transient compartmentalized diffusion with an average compartment size of 100-110 nm, transient confinement time of 8-10 ms, intracompartmental diffusion coefficient of 0.7-0.9 mu m2 s-1, and intercompartmental diffusion coefficient of 0.3-0.4 mu m2 s-1. The same analysis applied at the single-trajectory level identifies a complex variety of diffusion modes with 7-8% free, 13-14% confined, 40% transient compartmentalized, and 40% anomalous diffusion. Measurements with larger (& Oslash;40 nm) as compared to smaller (& Oslash;20 nm) gold nanoparticles are found to influence the diffusion rate and confinement strength, but not the underlying lipid diffusion modes. Using Monte Carlo simulations, these experimental results are explored in the wider context of relevant literature. This analysis paints a unifying picture of lipid diffusion on mammalian cell membranes transcending differences between experimental techniques.
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The authors greatly acknowledge the Engineering and Physical Sciences Research Council (EPSRC) and Medical Research Council (MRC) for supporting the DPhil project of FR within the Oxford-Nottingham Biomedical Imaging Centre of Doctoral Training (ONBI-CDT) (grant no. EP/L016052/1). Further, the authors acknowledge support by the EPA Cephalosporin Fund (Bio-ISCAT project), the MRC (grant no. MC_UU_12010/unit programs G0902418 and MC_UU_12025), the Wellcome Trust (grant no. 104924/14/Z/14 and Strategic Award 091911 (Micron)), MRC/BBSRC/EPSRC (grant no. MR/K01577X/1), the Wolfson Foundation (for initial funding of the Wolfson Imaging Centre Oxford), the John Fell Fund, the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; under research unit 1905 "Structure and function of the peroxisomal translocon", Germany's Excellence Strategy-EXC 2051-Project-ID 390713860; project number 316213987-SFB 1278; GRK M-M-M: GRK 2723/1-2023-ID 44711651; Instrument funding MINFLUX Jena INST 275_405_1; Instrument funding modular STED INST 1757/25-1 FUGG; GRK PhInt: GRK 3014/1), the State of Thuringia (TMWWDG), the Free State of Thuringia (TAB; AdvancedSTED/FGZ: 2018 FGI 0022; Advanced Flu-Spec/2020 FGZ: FGI 0031; Multi-XUV/2023 FGR 0054), the innovation program by the German BMWi (ZIM; project 16KN070967/Lab-on-a-chip SMARTIES), and the BMBF (Photonics Research Germany, FKZ: 13N15713/13N15717). The authors highlight that this research is integrated into the Leibniz Center for Photonics in Infection Research (LPI), which was initiated by Leibniz-IPHT, Leibniz-HKI, UKJ, and FSU Jena and is part of the BMBF national roadmap for research infrastructures. The authors would like to acknowledge the support of Dr. Erdinc Sezgin and Dr. Dilip Shrestha for their scientific advice, and the Kukura Group (Oxford University) for technical support in the initial phases of microscope development. Open Access funding enabled and organized by Projekt DEAL.
