The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Fluorescence imaging is one of the most versatile and widely-used tools in biology 1 . Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving 2, 3 , fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context 4, 5 . Here we report a solution to this problem, in the form of o ne-step n anoscale e xpansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis 6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ∼17 kDa protein calmodulin upon Ca 2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membraneassociated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.