ONE Microscopy

Super-resolution imaging has been applied to biological specimens for more than two decades. Nevertheless, it still falls short of describing the morphology of single molecules and the organization of molecular complexes, since most super-resolution microscopy implementations become error-prone below 25nm. Here we provide a simple and effective solution to this problem, one nanometer expansion (ONE) microscopy, generated by combining large-scale expansion microscopy with a detailed fluorescence fluctuation analysis. This approach was readily applied to molecular complexes, including synaptic vesicles, synaptic receptors, postsynaptic densities, microtubules, viral particles and alpha-synuclein aggregates, and to single molecules, as antibodies or the small calcium sensor calmodulin. ONE microscopy described molecular shapes and sizes, solved long-standing questions and revealed novel structures, implying that this is a promising approach for future applications. Its application should be especially facile, since ONE microscopy is compatible with conventional and even antiquated microscopes, and accommodates multiple expansion microscopy chemistries.