Abstract
Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 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 Ca2+ 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.
Competing Interest Statement
S.O.R. and F.O. are shareholders of NanoTag Biotechnologies GmbH. The remaining authors declare no competing interests. E.S.B. is an inventor on multiple patents related to expansion microscopy, and co-founder of a company working on commercial applications of expansion microscopy.
Footnotes
Shaib et al., 2022, has been updated to include many types of new data, all presented in new figures: -A demonstration of ONE feasibility in many laboratories in Germany and USA (Supp. Figs. 29 to 33) -Averaging analysis for single GABAa Receptors, to derive their protein shape in vitro, shown in Fig. 3, and supported by extensive new data (Supp. Figs. 12 and 13) -An averaging analysis of tubulin in vitro, to also derive its molecular shape (Supp. Fig. 15) -Averaging analysis for actin filaments in cells, again for the purpose of deriving their molecular shape (Fig. 4) -Analyses of ONE microscopy for post-expansion immunolabeling of PSD95 (Supp. Fig. 21), microtubules (Supp. Fig. 32) and bassoon (Supp. Fig. 33) -Analyses of ONE images in the sub-nanometer range (Supp. Fig. 24) -Quantifications of the SRRF precision, using DNA Origami nanorulers (Supp. Fig. 2) -An in-detail explanation of why ONE microscopy is able to provide molecular resolution (Supp. Fig. 3 and 4) -New analyses of single proteins, as eGFP (Supp. Fig. 8) -New analyses of synapses (Supp. Fig. 20) -Extensive FRC analyses of many samples (Supp. Figs. 23 and 28) -Technical updates, including structure analyses (Supp. Fig. 25), examples of bleaching curves (Supp. Fig. 7), drift correction (Supp. Fig. 14), analysis explanations and quantifications (Supp. Figs. 17 and 18), comparisons with current technologies (Supp. Fig. 22) Other updated elements: authors and author affiliations, main text, main figures, Materials and Methods, Extended Data Figures and Supplementary Figures.