Abstract
Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.
| Original language | English |
|---|---|
| Pages (from-to) | 1949-1956 |
| Number of pages | 25 |
| Journal | Nature Methods |
| Volume | 20 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - Dec 2023 |
| MoE publication type | A1 Journal article-refereed |
Funding
We thank J. Lippincott-Schwartz and C. Obara at Janelia Farm for their assistance with the dynamic ER-TIRF data and for reading the paper. This work was supported by the Gulbenkian Foundation (H.S.H., S. Coelho, D.M. and R.H.) and received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 101001332 to R.H. and D.M.), the European Commission through the Horizon Europe program (AI4LIFE project with grant agreement 101057970-AI4LIFE and RT-SuperES project with grant agreement 101099654-RT-SuperES to R.H.), the European Molecular Biology Organization (EMBO-2020-IG-4734 to R.H. and ALTF 499-2021 and Scientific Exchange Grant 9958 to H.S.H.), the Wellcome Trust (203276/Z/16/Z to R.H.), the Fundação para a Ciência e Tecnologia, Portugal (FCT fellowship CEECIND/01480/2021 to H.S.H., CEECIND/07466/2022 and PTDC/08248/2022 to S. Coelho), the Chan Zuckerberg Initiative Visual Proteomics Grant (vpi-0000000044 to R.H.), InnOValley Proof of Concept Fund (IOVPoC-2021-01 to S. Coelho) and National Health and Medical Research Council of Australia (no. APP1183588 to S. Coelho and J.G.). R.F.L. acknowledges the support of the MRC Skills Development Fellowship (MR/T027924/1). T.G. acknowledges the support by the European Union’s Horizon 2020 research and Innovation program under the Marie Sklodowska-Curie Grant Agreement No. 666003 and the Fondation pour la Recherche Médicale under the Fin de these 2020 program (FDT202001010813). UTechS PBI is part of the France-BioImaging infrastructure network and A. Salles and A.D. are supported by the French National Research Agency (ANR-10-INBS-04; Investments for the Future) and acknowledge support from ANR/FBI and the Région Ile-de-France (program ‘Domaine d’Intérêt Majeur-Malinf’) for the use of the Zeiss LSM 780 Elyra PS1 microscope. S. Culley acknowledges support from a Royal Society University Research Fellowship (URF\R1\211329). This study was supported by the Academy of Finland (G.J., 338537 and G.F., 332402), the Sigrid Juselius Foundation (G.J.), the Cancer Society of Finland (G.J.), the Åbo Akademi University Research Foundation (G.J., CoE CellMech), the Drug Discovery and Diagnostics strategic funding to Åbo Akademi University (G.J.) and by the InFLAMES Flagship Program of the Academy of Finland (decision no. 337531). The Cell Imaging and Cytometry Core facility (Turku Bioscience, University of Turku, Åbo Akademi University and Biocenter Finland) and Turku Bioimaging are acknowledged for services, instrumentation and expertise. C.L. acknowledges funding from the Agence National de la Recherche (ANR-20-CE13-0024). C.L. acknowledges the INP NCIS imaging facility and Nikon Center of Excellence for Neuro-NanoImaging for service and expertise, with funding from Excellence Initiative of Aix-Marseille University, A*MIDEX, a French ‘Investissements d’Avenir’ program (AMX-19-IET-002) through the Marseille Imaging and NeuroMarseille Institute. B.H. acknowledges funding from the Fondation pour la Recherche Médicale (DEI20151234398), the Agence National de la Recherche (ANR-19-CE42-0003-01), the LabEx Cell(n)Scale (ANR-11-LABX-0038, ANR-10-IDEX-0001-02), the Institut Curie, Agence pour la Recherche sur le Cancer (ARC Foundation), DIM ELICIT and from ITMO Cancer of Aviesan on funds administered by Inserm (grant no. 20CP092-00). B.H. recognizes the support of France-BioImaging infrastructure grant ANR-10-INBS-04 (Investments for the Future).