Super-resolution microscopy enables imaging with spatial resolution beyond the diffraction limit. Although the range of available techniques is wide, diverse and ever-growing, they all share the potential to provide insights into the workings of proteins, cells and organisms. Each technique, however, comes with its own strengths and limitations, making them suitable for specific applications depending on the demands for spatial resolution, imaging speed, field of view and sample preparation.

In this Focus issue, we explore the state of the art in selected areas of fluorescence super-resolution microscopy. This includes a Review by Jonas Ries and colleagues from the University of Vienna, which highlights recent advancements and future directions for MINimal fluorescence photon FLUxes (MINFLUX) microscopy, and another Review authored by Jörg Enderlein’s group at Georg August University, focusing on super-resolution optical fluctuation imaging (SOFI). We also feature one Q&A with Markus Sauer, from the University of Würzburg, who shares his views about the current capabilities and remaining challenges of SR microscopy for biological applications. In another Q&A with Stefan Hell, from the Max Planck Institute for Multidisciplinary Sciences, we discuss key milestones for the field, as well as the current capabilities and future perspectives for MINFLUX.

Our aim with this Focus is to highlight key advances that have driven substantial progress in fluorescence super-resolution microscopy in the last decade or so. When the 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for the invention of PALM/STORM and STED, spatial resolution was only a few tens of nanometres and imaging was slow, making it impossible to visualize the nanoscale dynamics of proteins in live cells. Fast-forward ten years, and techniques like MINFLUX can localize proteins in their native state with sub-nanometre precision and a temporal resolution of 5 milliseconds. Achieving super-resolution over extended fields of view has also always been challenging, especially for point-scanning techniques. Today, wide-field techniques such as SOFI and SIM enable imaging over a few millimetres squared within a few minutes and with a spatial resolution on the order of 100 nm.

Reflecting on this journey shows the astonishing progress at the conceptual and technical level, as well as the growth of practical applications. Key innovations have included novel optical principles, increasingly sensitive instrumentation, better fluorophore design, refined labelling protocols, and advanced computational algorithms for image reconstruction and post-acquisition enhancement. Among these, we especially appreciate the value of optics-driven progress, most notably in the principles that have been developed to overcome Abbe's diffraction limit. These range from STED and its derivatives, which allow precise control of fluorescence excitation and depletion to confine emission below the diffraction limit, to PALM/STORM methods that isolate individual fluorophores through temporal activation and ON/OFF switching dynamics — the breakthroughs that earned the 2014 Nobel Prize in Chemistry. Progress also extends to structured illumination microscopy, where high-resolution spatial information is extracted by engineering illumination patterns coupled with ever-advancing reconstruction algorithms, and to SOFI-inspired approaches, which analyse the temporal fluctuations of fluorophores to push beyond the diffraction limit. Other recent developments, such as MINSTED and MINFLUX, also enable impressive imaging speed and resolution by scanning a doughnut-shaped excitation beam around the fluorophore to be localized.

What’s next then?

First, it is becoming important to explore how such high levels of precision can be reliably translated to biomedical imaging settings while coping with the complexity inherent to the biological world. For this reason, optics-driven advancements in super-resolution microscopy must involve close collaboration with biologists, to understand their needs and what questions need answers. Meaningful development is often initiated, and then driven, by asking the right questions and finding the right problem to solve, aligning efforts with real needs. Collaboration with (bio)chemists is also important to make sure labelling workflows are feasible and reliable.

Second, as spatial resolution approaches the size of fluorophores themselves, we enter what is often referred to as nanoscopy. Although this opens up exciting possibilities for studying proteins and potentially their conformational changes with unprecedented detail, it also comes with challenges. Labelling samples at such an extreme scale is difficult, as existing methods struggle to attach fluorescent tags just a few nanometres apart within cells. We also need to deal with the different photochemistry of two fluorophores spaced by less than 10 nm, as their photo-switching behaviour can be altered, reducing the probability of accurate localization. Importantly, this challenge is shared across multiple techniques, showing the importance of finding solutions.

Finally, the ability to image live cells over long periods of time is highly valued when it comes to studying dynamical processes in real time. However, super-resolution microscopy works best with fixed samples. Live cells are mobile, thus demanding careful optimization of the trade-off between spatial resolution, imaging speed and field of view. Hours-long imaging also raises challenges of phototoxicity and photobleaching, and it highlights the need for new fluorescent probes that are both stable and non-invasive over long periods.

Overall, this Focus issue attempts to capture a selection of recent developments and spark discussions about new opportunities and outstanding challenges to increase our understanding of the biological world. Through synergistic efforts from optics, biology and biochemistry, super-resolution microscopy is poised to keep developing, gain wider adoption and provide solutions to real-world biomedical challenges that can tangibly improve healthcare and clinical outcomes.