Visualizing proteins by expansion microscopy

Abstract

Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image 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 could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an 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.

An optimized approach to study sub-sarcomere structure utilizing super-resolution microscopy with secondary VHH nanobodies

“…We suggest that use of this approach with models of muscle weakness holds potential to help identify potential changes in protein localization and potential disruptions in sub-sarcomere structure that could contribute to functional declines. We note that our approach is not the first to utilize advanced imaging modalities to study skeletal muscle structure (Radermacher et al ., 1994; Blanc et al ., 2000; Soeller & Baddeley, 2013; Glancy et al ., 2014; Sun et al ., 2014; Jayasinghe et al ., 2014, 2015, 2018; Agarwal & Machá, 2016; York & Zheng, 2017; Gemmink et al ., 2018; Yi et al ., 2019; Ghosh et al ., 2019; Szikora et al ., 2020; Daneshparvar et al ., 2020; Oda & Yanagisawa, 2020; Hofemeier et al ., 2021; Rahmanseresht et al ., 2021; van der Pijl et al ., 2021 a ; Wang et al ., 2021; Melville et al ., 2022; Rimoli et al ., 2022; Skorska et al ., 2022; Schueder et al ., 2022; McMillan & Scarff, 2022; Shaib et al , 2022). Many of these approaches implement single molecule super-resolution microscopy techniques, such as stochastic optical reconstruction microscopy (STORM).…”

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…The earliest example of this was the combination of the original 4-fold expanding expansion microscopy protocol with STED microscopy, for a combined resolution below 10 nm . Almost all major optical super-resolution modalities have now successfully been combined with various expansion microscopy techniques. − ,,− …”

“…On the face of it, isolated cell components, such as proteins, protein complexes, , organelles such as nuclei, , and other components such as chromosomes, ,, may seem more amenable to expansion even than cell culture. After all, they pose even lower diffusion barriers and mechanical resistance.…”

Unclearing Microscopy

“…Analogous to electron microscopy (EM), subcellular compartments can now be imaged in their entirety, without the need for specific labels and with a lateral spatial resolution of ~15 nm. Our pan-staining concept has been since adapted by many labs to examine contextual spatial information in a multitude of biological systems, and at different expansion levels, demonstrating the wide utility of this contrasting approach (Nijenhuis et al, 2021;Sim et al, 2021;Klimas et al, 2021;Damstra et al, 2022;Laporte et al, 2022;Shaib et al, 2022;Hinterndorfer et al, 2022;Rashpa and Brochet, 2022;Pacheco et al, 2021;Suen et al 2022).…”