For the longest time, light microscopy was bound by the classical diffraction limit of visible light, which prevented scientists from clearly resolving cellular structures smaller than 200 nanometers.1 However, in the early 1990s, scientists conceived the first concepts to overcome this barrier, refining methods to develop super-resolution imaging techniques. Over the past three decades, the super-resolution microscopy field has evolved to allow researchers to observe individual proteins traveling in real time, map complex three-dimensional organoids, and accelerate drug discovery. In celebration of The Scientist’s 40th anniversary, we dug through our archives to highlight our coverage of the super-resolution microscopy field through the years.
2004: The First Super-Resolution Microscope Hits the Market
After overcoming optical diffraction limits, companies started manufacturing super-resolution imaging instruments, but their user base remained restricted to those who invented the machines. But in 2004, Leica announced a new super-resolution microscope and made it available to some partners, including academic institutions. Making 4Pi microscopy available to the broader scientific marketplace helped move advanced imaging out of physics laboratories into the hands of life science researchers who could peek into cellular architecture at an unprecedented level.
2010: The Fundamental Concepts and Methods Behind Super-Resolution Microscopy
What principles of foundational physics helped researchers break the 200-nanometer diffraction barrier? What are some of the methods helping researchers view cells at a high resolution? As super-resolution microscopy became more accessible, researchers developed new techniques. In 2010, scientists were already exploring the concepts and practical applications of super-resolution imaging techniques such as Photoactivated Localization Microscopy (PALM), Stimulated Emission Depletion (STED) microscopy, and Stochastic Optical Reconstruction Microscopy (STORM).
2013: Tweaks in GFP Help Capture Cellular Dynamics
Dynamic imaging of live cells is crucial to understanding cellular processes, but scientists remained limited in their ability to do so due to technical challenges. While STED microscopy could help visualize small organelles within cells, the technique required bright lasers that could damage living cells. Researchers led by Stefan Hell and Stefan Jakobs at the Max Planck Institute for Biophysical Chemistry, mutated a few amino acids in green fluorescent protein (GFP) to produce enhanced GFP, rendering a more resilient variant of the fluorophore. The engineered probe allowed techniques like Reversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) with less phototoxicity than previous fluorophores allowed.
2014: Nobel Prize for Super-Resolution Microscopy
The crowning achievement of the super-resolution microscopy revolution came in 2014, when Eric Betzig, Hell, and William Moerner won the 2014 Nobel Prize in Chemistry. The award honored their pioneering contributions to developing super-resolution fluorescence microscopy, which broke the theoretical limit of microscopic resolution imposed by the wavelength of light. The Nobel Foundation announced that the three were being honored for bringing “optical microscopy into the nanodimension,” enabling scientists to “study living cells in the tiniest molecular detail.” In an interview with The Scientist, Moerner shared events that led to this historic milestone.
2015: Super-Resolution Microscopy Goes Live
While super-resolution microscopy offered an unprecedented window into cellular architecture, the technique remained restricted to fixed, dead samples. By modifying structured illumination microscopy (SIM) to improve its resolution, Betzig and his colleagues were able to visualize dynamic cellular processes including endocytosis and cytoskeletal remodeling. This innovation represented a profound paradigm shift within the bioimaging community.
2022: JeWell Chips Allow Super-Resolution Imaging of 3D Organoids
Even as the organoid field rapidly advanced, with scientists generating different types of organoids from distinct cells, imaging these 3D structures remained tricky. University of Bordeaux physicist Jean-Baptiste Sibarita and his team developed a technique to obtain 3D images of live cells, but applying this method to organoids was challenging and slow. By joining forces with National University of Singapore cell biologist Anne Beghin, Sibarita designed microfabricated organoid culture chips termed JeWells to image organoids in a high-throughput manner. Using their approach, the researchers imaged a single organoid in seven seconds, and roughly 300 organoids in an hour, solving a practical hardware and scaling problem of applying super-resolution microscopy to organoids.
2023: Fluorescence Microscopy Tracks Motor Proteins Along Cellular Highways
Hell and his team developed a type of fluorescence microscopy that helped them track proteins at nanometer resolution in 2016. In 2023, the researchers improved upon this to obtain enhanced spatiotemporal resolution. With the updated technique, the researchers fluorescently tagged and monitored a motor protein called kinesin as it walked down the length of a microtubule. The technical modification to high-resolution fluorescence microscopy pushed the boundaries of kinetic tracking, unlocking the ability to monitor motor proteins’ highly precise, nanoscale steps and structural movement.
2023: Swelling Cells Enabled Visualization of Cellular Architecture with Simple Microscopes
In 2015, scientists developed a technique called expansion microscopy. Instead of adjusting microscope settings, this technique physically inflates and stains biological samples, making them large enough to observe using a simple microscope. Yale University cell biologists Joerg Bewersdorf and Ons M’Saad further developed the technique, enabling cells to be seen with the naked eye. Their technique, which they dubbed “Unclearing Microscopy,” could eventually allow scientists to do super-resolution microscopy without state-of-the-art microscopes, potentially making microscopy research more broadly accessible.
2025: Super-Resolution Techniques Reveal How Antibodies Target Tumor Cells
Markus Sauer, a biophysicist at the University of Würzburg, and his colleagues found a way to visualize the molecular interactions between therapeutic monoclonal antibodies (mAbs) and their target receptors on tumor cells. They combined a technique called DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) with lattice light-sheet microscopy to develop a high-speed 3D super-resolution platform that significantly reduced imaging times. Using this method, Sauer and his team found that mAbs grouped in different classes triggered similar cellular changes, challenging conventional mAb classification.
- Prakash K, et al. Super-resolution microscopy: A brief history and new avenues. Philos Trans A Math Phys Eng Sci. 2022;380(2220):20210110.
