Technology

NanoJ-SRRF

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GitHub:
- HenriquesLab/NanoJ-SRRF

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Publication: Gustafsson et al. Nature Communications 2016
- 619

Super-resolution radial fluctuations (SRRF) is an analytical method that produces super-resolution images of fixed and live cells from data acquired on standard widefield, TIRF or confocal microscopes with conventional fluorophores (Gustafsson et al., 2016, Nature Communications; Culley et al., 2018, International Journal of Biochemistry & Cell Biology). The method works with standard dyes and fluorescent proteins at low laser power, and it does not localise single molecules.

SRRF treats each raw frame as a set of point sources blurred by the microscope's point spread function. It first magnifies each frame onto a finer pixel grid. For every point of this grid, it then computes the radiality: how strongly the local intensity gradients converge on that point. A fluorophore produces gradients that point towards its centre, so the radiality map shows a sharp peak at each emitter, much narrower than the point spread function. Radiality depends on the symmetry of the local gradients and hardly at all on brightness, so dim and bright emitters give peaks of similar width. The method weights the radiality by the local intensity and gradient magnitude to suppress peaks that come from noise.

SRRF then analyses how the radiality at each point changes across the frame sequence. Fluorophores blink and fluctuate, and the radiality peaks they produce stay at fixed positions from frame to frame, while noise peaks appear at random. Temporal statistics of the radiality stack, such as the mean, auto-correlations or higher-order cumulants, reinforce the correlated signal at real emitters and cancel the uncorrelated noise. The width of the radiality peaks sets the resolution of the final image.

Because it works from fluctuations and from gradient symmetry, SRRF handles fluorophore densities from sparse to dense. At low density its precision approaches that of single-molecule localisation; at densities too high for localisation it still resolves structures around 100–150 nm apart. It needs only low illumination, so it can follow living cells for long periods with little phototoxicity. From as few as 100 raw frames SRRF can produce about one super-resolution frame per second, fast enough to follow nanoscale dynamics in living cells.

NanoJ-SRRF is the open-source ImageJ/Fiji implementation of the method, with GPU acceleration. Its successors, eSRRF and the Python implementation in NanoPyx, build on the same principles.

Publications featuring NanoJ-SRRF

Expansion and fluctuations-enhanced microscopy for nanoscale molecular profiling of cells and tissues Dominik Kylies, Hannah S. Heil, Arturo G. Vesga, Mario Del Rosario, Maria Schwerk, Malte Kuehl, Milagros N. Wong, Victor G. Puelles, Ricardo Henriques Paper published in Nature Protocols, July 2025 Technologies: NanoJ (), NanoJ-eSRRF (), NanoJ-SQUIRREL (), NanoJ-SRRF (), NanoPyx () and Nuclear-Pores as references Funded by: Chan Zuckerberg Initiative (CZI), The Kavli Foundation, and The Wellcome Trust, CZI, EMBO, ERC, FCT, H2021 and H2022 DOI: 10.1038/s41596-025-01178-0

Nanoscale imaging of biological systems via expansion and super-resolution microscopy Daria Aristova, Dominik Kylies, Mario Del Rosario, Hannah S. Heil, Maria Schwerk, Malte Kuehl, Milagros N. Wong, Ricardo Henriques, Victor G. Puelles Paper published in Applied Physics Reviews, April 2025 Technologies: NanoJ-eSRRF () and NanoJ-SRRF () Funded by: Chan Zuckerberg Initiative (CZI), The Kavli Foundation, and The Wellcome Trust, CZI, EMBO, ERC, FCT, H2021 and H2022 DOI: 10.1063/5.0240464

Harnessing artificial intelligence to reduce phototoxicity in live imaging Estibaliz Gómez-de-Mariscal, Mario Del Rosario, Joanna W. Pylvänäinen, Guillaume Jacquemet, Ricardo Henriques Perspective published in Journal of Cell Science, February 2024 Technologies: BioImage Model Zoo (), CARE (), DeepBacs (), NanoJ-eSRRF (), NanoJ-SQUIRREL (), NanoJ-SRRF () and ZeroCostDL4Mic () Funded by: CZI, EMBO, ERC, H2021 and H2022 DOI: 10.1242/jcs.261545

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation Romain F. Laine, Hannah S. Heil, Simao Coelho, Jonathon Nixon-Abell, Angélique Jimenez, Theresa Wiesner, Damián Martínez, Tommaso Galgani, Louise Régnier, Aki Stubb, Gautier Follain, Samantha Webster, Jesse Goyette, Aurelien Dauphin, Audrey Salles, Siân Culley, Guillaume Jacquemet, Bassam Hajj, Christophe Leterrier, Ricardo Henriques Paper published in Nature Methods, November 2023 Technologies: CARE (), NanoJ (), NanoJ-eSRRF (), NanoJ-SQUIRREL (), NanoJ-SRRF () and Nuclear-Pores as references Funded by: CZI, EMBO, ERC, FCT, H2021, H2022, InnOValley and Wellcome Trust News: Photonics.com, The Science Times, Optics.org and Phys.org Blogs: Springer Nature Protocols and Methods Community DOI: 10.1038/s41592-023-02057-w

The LEGO® brick road to open science and biotechnology Etienne Boulter, Julien Colombelli, Ricardo Henriques, Chloé C. Féral Review published in Trends in Biotechnology, March 2022 Technologies: NanoJ (), NanoJ-Fluidics () and NanoJ-SRRF () Funded by: EMBO, ERC and Wellcome Trust DOI: 10.1016/j.tibtech.2022.02.003

Democratising deep learning for microscopy with ZeroCostDL4Mic Lucas von Chamier, Romain F. Laine, Johanna Jukkala, Christoph Spahn, Daniel Krentzel, Elias Nehme, Martina Lerche, Sara Hernández-Pérez, Pieta K. Mattila, Eleni Karinou, Séamus Holden, Ahmet Can Solak, Alexander Krull, Tim-Oliver Buchholz, Martin L. Jones, Loïc A. Royer, Christophe Leterrier, Yoav Shechtman, Florian Jug, Mike Heilemann, Guillaume Jacquemet, Ricardo Henriques Paper published in Nature Communications, April 2021 Technologies: CARE (), NanoJ (), NanoJ-SQUIRREL (), NanoJ-SRRF () and ZeroCostDL4Mic () Funded by: EMBO, ERC and Wellcome Trust News: AZO Life Sciences, Drug Target Review, Nanotechnology Now and The Medical News Blogs: Microbiome Digest - Bik's Picks DOI: 10.1038/s41467-021-22518-0

Closed mitosis requires local disassembly of the nuclear envelope Gautam Dey, Siân Culley, Scott Curran, Uwe Schmidt, Ricardo Henriques, Wanda Kukulski, Buzz Baum Paper published in Nature, August 2020 Technologies: CARE (), NanoJ (), NanoJ-SRRF () and Nuclear-Pores as references Funded by: BBSRC and Wellcome Trust News: Nature Asia DOI: 10.1038/s41586-020-2648-3

The proteasome controls ESCRT-III–mediated cell division in an archaeon Gabriel Tarrason Risa, Fredrik Hurtig, Sian Bray, Anne E. Hafner, Lena Harker-Kirschneck, Peter Faull, Colin Davis, Dimitra Papatziamou, Delyan R. Mutavchiev, Catherine Fan, Leticia Meneguello, Andre Arashiro Pulschen, Gautam Dey, Siân Culley, Mairi Kilkenny, Diorge P. Souza, Luca Pellegrini, Robertus A. M. de Bruin, Ricardo Henriques, Ambrosius P. Snijders, Anđela Šarić, Ann-Christin Lindås, Nicholas P. Robinson, Buzz Baum Paper published in Science, August 2020 Technologies: NanoJ () and NanoJ-SRRF () News: NRK, Scitech Daily, NCYT - Noticias de la Ciencia y la Technologia and UPI.com DOI: 10.1126/science.aaz2532

The cell biologist's guide to super-resolution microscopy Guillaume Jacquemet, Alexandre F. Carisey, Hellyeh Hamidi, Ricardo Henriques, Christophe Leterrier Review published in Journal of Cell Science, June 2020 Technologies: CARE (), NanoJ (), NanoJ-Fluidics (), NanoJ-SRRF () and Nuclear-Pores as references Funded by: BBSRC and Wellcome Trust News: ScienMag and EurekAlert! DOI: 10.1242/jcs.240713

Between life and death: strategies to reduce phototoxicity in super-resolution microscopy Kalina L Tosheva, Yue Yuan, Pedro Matos Pereira, Siân Culley, Ricardo Henriques Review published in Journal of Physics D: Applied Physics, January 2020 Technologies: CARE (), NanoJ (), NanoJ-Fluidics () and NanoJ-SRRF () Funded by: BBSRC and Wellcome Trust DOI: 10.1088/1361-6463/ab6b95

Super‐beacons: Open‐source probes with spontaneous tuneable blinking compatible with live‐cell super‐resolution microscopy Pedro M Pereira, Nils Gustafsson, Mark Marsh, Musa M Mhlanga, Ricardo Henriques Paper published in Traffic, January 2020 Technologies: NanoJ (), NanoJ-Fluidics (), NanoJ-SQUIRREL (), NanoJ-SRRF () and Super-Beacons Funded by: BBSRC and Wellcome Trust DOI: 10.1111/tra.12728

Fluctuation-based super-resolution traction force microscopy Aki Stubb, Romain F Laine, Mitro Miihkinen, Hellyeh Hamidi, Camilo Guzmán, Ricardo Henriques, Guillaume Jacquemet, Johanna Ivaska Paper published in Nano letters, January 2020 Technologies: FBSR-TFM, NanoJ (), NanoJ-SQUIRREL () and NanoJ-SRRF () Funded by: BBSRC DOI: 10.1021/acs.nanolett.9b04083

Nuclear pores as versatile reference standards for quantitative superresolution microscopy Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieśliński, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya Kasuba, Sarah Janice Hoerner, Yiming Li, Yu-Le Wu, Markus Mund, Ulf Matti, Pedro Matos Pereira, Ricardo Henriques, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jan Ellenberg, Jonas Ries Paper published in Nature Methods, September 2019 Technologies: CARE (), NanoJ (), NanoJ-SQUIRREL (), NanoJ-SRRF () and Nuclear-Pores as references Funded by: BBSRC and Wellcome Trust News: Mirage News DOI: 10.1038/s41592-019-0574-9

Artificial intelligence for microscopy: what you should know Lucas von Chamier, Romain F. Laine, Ricardo Henriques Review published in Biochemical Society Transactions, July 2019 Technologies: CARE (), NanoJ (), NanoJ-Fluidics (), NanoJ-SQUIRREL () and NanoJ-SRRF () Funded by: BBSRC and Wellcome Trust News: Azooptics.com DOI: 10.1042/bst20180391

Automating multimodal microscopy with NanoJ-Fluidics Pedro Almada, Pedro M. Pereira, Siân Culley, Ghislaine Caillol, Fanny Boroni-Rueda, Christina L. Dix, Guillaume Charras, Buzz Baum, Romain F. Laine, Christophe Leterrier, Ricardo Henriques Paper published in Nature Communications, March 2019 Technologies: NanoJ (), NanoJ-Fluidics (), NanoJ-SQUIRREL (), NanoJ-SRRF () and NanoJ-VirusMapper Funded by: BBSRC and Wellcome Trust News: Technology Times, MSN, DNYUZ and Express Informer DOI: 10.1038/s41467-019-09231-9

NanoJ: a high-performance open-source super-resolution microscopy toolbox Romain F Laine, Kalina L Tosheva, Nils Gustafsson, Robert D M Gray, Pedro Almada, David Albrecht, Gabriel T Risa, Fredrik Hurtig, Ann-Christin Lindås, Buzz Baum, Jason Mercer, Christophe Leterrier, Pedro M Pereira, Siân Culley, Ricardo Henriques Paper published in Journal of Physics D: Applied Physics, January 2019 Technologies: CARE (), NanoJ (), NanoJ-SQUIRREL (), NanoJ-SRRF (), NanoJ-VirusMapper and QuickPALM Funded by: BBSRC and Wellcome Trust DOI: 10.1088/1361-6463/ab0261

Real time multi-modal super-resolution microscopy through Super-Resolution Radial Fluctuations (SRRF-Stream) Justin Cooper, Mark Browne, Hugh Gribben, Martin Catney, Colin Coates, Alan Mullan, Geraint Wilde, Ricardo Henriques Paper published in Single molecule spectroscopy and superresolution imaging XII, January 2019 Technologies: NanoJ-SRRF () DOI: 10.1117/12.2510761

Content-aware image restoration: pushing the limits of fluorescence microscopy Martin Weigert, Uwe Schmidt, Tobias Boothe, Andreas Müller, Alexandr Dibrov, Akanksha Jain, Benjamin Wilhelm, Deborah Schmidt, Coleman Broaddus, Siân Culley, Mauricio Rocha-Martins, Fabián Segovia-Miranda, Caren Norden, Ricardo Henriques, Marino Zerial, Michele Solimena, Jochen Rink, Pavel Tomancak, Loic Royer, Florian Jug, Eugene W. Myers Paper published in Nature Methods, November 2018 Technologies: CARE (), NanoJ-SQUIRREL () and NanoJ-SRRF () Funded by: BBSRC and Wellcome Trust News: Technology Networks, VBIO, Innovations Report and Informationsdienst Wissenschaft DOI: 10.1038/s41592-018-0216-7

Heterogeneous localisation of membrane proteins in Staphylococcus aureus Felix Weihs, Katarzyna Wacnik, Robert D. Turner, Siân Culley, Ricardo Henriques, Simon J. Foster Paper published in Scientific Reports, February 2018 Technologies: NanoJ-SRRF () Funded by: BBSRC and Wellcome Trust DOI: 10.1038/s41598-018-21750-x

Quantitative mapping and minimization of super-resolution optical imaging artifacts Siân Culley, David Albrecht, Caron Jacobs, Pedro Matos Pereira, Christophe Leterrier, Jason Mercer, Ricardo Henriques Paper published in Nature Methods, February 2018 Technologies: NanoJ-SQUIRREL (), NanoJ-SRRF () and QuickPALM Funded by: BBSRC and Wellcome Trust News: physicsworld.com DOI: 10.1038/nmeth.4605

SRRF: Universal live-cell super-resolution microscopy Siân Culley, Kalina L Tosheva, Pedro Matos Pereira, Ricardo Henriques Paper published in The international journal of biochemistry & cell biology, January 2018 Technologies: NanoJ-SQUIRREL () and NanoJ-SRRF () Funded by: BBSRC and Wellcome Trust DOI: 10.1016/j.biocel.2018.05.014

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations Nils Gustafsson, Siân Culley, George Ashdown, Dylan M. Owen, Pedro Matos Pereira, Ricardo Henriques Paper published in Nature Communications, August 2016 Technologies: NanoJ-SRRF () and QuickPALM Funded by: BBSRC News: Azom.com DOI: 10.1038/ncomms12471

Funding contributing to NanoJ-SRRF

VirusAwareScopes: Machine Learning-Driven Adaptive Microscopy for Long-Term Viral Infection Studies Ricardo Henriques Alias: VirusAwareScopes Funded by: La Caixa Foundation - Health Research Duration: November 2025 - October 2028 Publications: 4

3D Nanoscope: a highly accessible, high-performance device for live cell nanoscopy Arturo G. Vesga Alias: 3DNanoScope4All Funded by: Marie Curie - HORIZON TMA MSCA Postdoctoral Fellowships Duration: March 2024 - February 2026

Sub-cellular Metabolic Compartmentalization During Oocyte Development Zita Carvalho dos Santos, Ricardo Henriques, Jorge Carvalho Funded by: CZI - Measuring Metabolism Across Scales Duration: January 2024 - December 2026

Real-Time high-content Super-Resolution Imaging of ES Cell States Eran Meshorer, Ricardo Henriques, Anna Kreshuk, Sandrine Lévêque-Fort, Nicolas Bourg, Genevieve Almouzni Alias: RT-SuperES Funded by: H2022 - EIC Pathfinder Open Duration: July 2023 - June 2027 Publications: 21

How does membrane topology influence T-cell activation and HIV infection? Simao Coelho Funded by: FCT - Exploratory Research Projects Duration: March 2023 - September 2024

Cutting-edge super-resolution image analysis in napari through NanoJ Bruno Saraiva, Ricardo Henriques Funded by: CZI - Applications - napari Plugin Foundations grants Duration: January 2023 - December 2023 Publications: 3

Artificial Intelligence for Image Data Analysis in the Life Sciences Anna Kreshuk, Florian Jug, Ricardo Henriques, Wei Ouyang, Arrate Muñoz-Barrutia, Emma Lundberg, Matthew Hartley Alias: AI4Life Funded by: H2021 - INFRA Duration: September 2022 - August 2025 Publications: 21

VP-CLEM-KIT: a pipeline for democratising volumetric visual proteomics Lucy Collinson, Ricardo Henriques, Paul French Funded by: CZI - Visual Proteomics Imaging Duration: December 2021 - June 2024 Publications: 19

Optial Biology PhD programme Michael Hausser, Ricardo Henriques, Antonella Riccio Funded by: Wellcome Trust - 4-year PhD Programme in Science Duration: August 2021 - August 2025

Enabling Live-Cell 4D Super-Resolution Microscopy Guided by Artificial Intelligence Ricardo Henriques Alias: SelfDriving4DSR Funded by: ERC - Consolidator Duration: July 2021 - June 2027 Publications: 40

Unveiling live-cell viral replication at the nanoscale Ricardo Henriques Funded by: EMBO - Installation Grant Duration: January 2021 - December 2026 Publications: 32