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Resolution in super-resolution microscopy — definition, trade-offs and perspectives

Nature Reviews Molecular Cell Biology volume 25, pages 677–682 (2024)Cite this article

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Super-resolution microscopy (SRM) is gaining popularity in biosciences; however, claims about optical resolution are contested and often misleading. In this Viewpoint, experts share their views on resolution and common trade-offs, such as labelling and post-processing, aiming to clarify them for biologists and facilitate deeper understanding and best use of SRM.

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Author information

Authors and Affiliations

  1. Division of Molecular Pathology, The Institute of Cancer Research, London, UK

    Kirti Prakash

  2. The Royal Marsden NHS Foundation Trust, London, UK

    Kirti Prakash

  3. Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand

    David Baddeley

  4. Institute of Applied Optics and Biophysics and Abbe Center of Photonics, Friedrich-Schiller-University Jena, Jena, Germany

    Christian Eggeling

  5. Leibniz Institute of Photonic Technology, Jena, Germany

    Christian Eggeling & Rainer Heintzmann

  6. Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Reto Fiolka

  7. Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University Jena, Jena, Germany

    Rainer Heintzmann

  8. Laboratory of Experimental Biophysics, School of Basic Sciences, Institute of Physics, Interfaculty Institute of Bioengineering, EPFL SB-LEB, Lausanne, Switzerland

    Suliana Manley

  9. Laboratory of Nanoscale Biology, School of Engineering, Institute of Bioengineering, EPFL STI IBI-STI LBEN, Lausanne, Switzerland

    Aleksandra Radenovic

  10. Delft Center for Systems and Control, Faculty of Mechanical, Maritime, and Materials Engineering, Technische Universiteit Delft, Delft, The Netherlands

    Carlas Smith

  11. Janelia Research Campus, Howard Hughes Medical Institute (HHMI), Ashburn, VA, USA

    Hari Shroff

  12. Department of Biochemistry, University of Oxford, Oxford, UK

    Lothar Schermelleh

Authors
  1. Kirti Prakash
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  2. David Baddeley
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  3. Christian Eggeling
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  4. Reto Fiolka
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  5. Rainer Heintzmann
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  6. Suliana Manley
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  7. Aleksandra Radenovic
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  8. Carlas Smith
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  9. Hari Shroff
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  10. Lothar Schermelleh
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Contributions

Kirti Prakash has degrees in computer science and biology. His expertise lies in single-molecule super-resolution microscopy, mathematical modelling and machine learning. He is dedicated to developing pioneering tools for advanced microscopy, with a research focus on epigenetics and chromatin structure.

David Baddeley is trained as a physicist and started working on super-resolution imaging problems in his doctorate. He has expertise in a range of methodologies including SMLM and a particular interest in the quantitative analysis of super-resolution data.

Christian Eggeling holds a degree in physics and, after gaining industry experience in single-molecule microscopy, joined the super-resolution microscopy group of Stefan Hell. He now leads an independent group advancing super-resolution microscopy and spectroscopy, focusing on molecular diffusion dynamics studies of cell membranes. He also manages a microscope facility.

Reto Fiolka, originally trained as a mechanical engineer in computational fluid dynamics, switched focus to optical microscopy during his doctorate. His laboratory is dedicated to developing new imaging techniques for biomedical research to be applied in 3D environments, ex vivo and in vivo, offering improved spatiotemporal resolution and multi-scale capabilities.

Rainer Heintzmann studied physics and computer science and develops super-resolution fluorescence microscopy methods such as linear and nonlinear structured illumination, pointillism and image inversion interferometry. He has a strong interest in computational optics and inverse problems such as deconvolution and in extracting multidimensional information from biological structures.

Suliana Manley studied physics and mathematics and became fascinated by complex biological systems. Her group studies the biophysical principles of organelle structure and dynamics and develops smart, automated and multi-modal microscopy methods. Their work is enriched by interplay between fundamental discovery and microscopy development.

Aleksandra Radenovic studied physics and is an expert in single-molecule biophysics and nanofluidics. Her research focuses on developing biosensors and optical imaging techniques for observing individual molecules and complexes, enhancing our understanding of their behaviour in various environments.

Carlas Smith studied aerospace engineering and applied physics. He now leads a group focusing on developing advanced computational microscopy techniques and the combination of opto-mechatronics and information processing algorithms, particularly for super-resolution imaging.

Hari Shroff has degrees in bioengineering and biophysics and entered the microscopy field as a postdoctoral researcher working on super-resolution imaging. He now leads a group focused on developing new optical and computational methods that offer the ability to interrogate biological structure and function across diverse spatiotemporal scales.

Lothar Schermelleh is a trained cell biologist who has specialized in the biological application of super-resolution structured illumination microscopy. His research focuses on studying chromatin organization and functional nuclear architecture. To this end, his group developed fluorescent labelling protocols and software tools for image data quality control.

Corresponding authors

Correspondence to Kirti Prakash, David Baddeley, Christian Eggeling, Reto Fiolka, Rainer Heintzmann, Suliana Manley, Aleksandra Radenovic, Carlas Smith, Hari Shroff or Lothar Schermelleh.

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Prakash, K., Baddeley, D., Eggeling, C. et al. Resolution in super-resolution microscopy — definition, trade-offs and perspectives. Nat Rev Mol Cell Biol 25, 677–682 (2024). https://doi.org/10.1038/s41580-024-00755-7

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