Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies (original) (raw)
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11-fold Expansion Microscopy with Universal Molecular Retention Using Magnify
Microscopy and Microanalysis
Increased accessibility to nanoscale imaging techniques to interrogate the spatial arrangement of biomolecules from the nanoscale to tissue level would greatly improve understanding of biological systems. Expansion microscopy (ExM) [1, 2] is a recently developed imaging technique that relies on physically and isotropically expanding the specimen, rather than relying on optical techniques to improve resolution. This is achieved by chemically embedding the tissue in a water-swellable polymer, mechanically homogenizing it, and physically expanding the tissue-gel hybrid [3]. This then allows the gel-anchored tissue biomolecules that were once previously overlapping within the diffraction limit to be physically separated, allowing them to be imaged using a conventional diffraction limited optical microscope. Although previous ExM protocols have been developed to perform nanoscale imaging proteins [4-11], nucleic acids [8, 12-14], and lipids[9, 10, 15], and new gel chemistries have been developed to achieve 10-fold or greater expansion with single[16, 17] or iterative [7, 18] expansion steps, no single protocol has been developed that achieves 10-fold or greater expansion while simultaneously conserving multiple biomolecules across a broad array of tissue types. To overcome this, we developed Magnify [19], a new ExM framework with a hydrogel formula that retains multiple biomolecule classes, including proteins, nucleic acids, and lipids that can be labeled post-expansion in a broad range of biological specimens. Here, tissues preserved with commonly used fixation methods can be expanded by up to ∼11-fold, resulting in an effective resolution of ∼25 nm for an ∼280-nm diffraction-limited 1.15 NA objective lens (∼280/11) on a conventional spinning disk confocal microscope (Fig. 1). When combined with Super-resolution Optical Fluctuation Imaging (SOFI),[20] a computational postprocessing method that relies on the independent temporal fluctuations of fluorophores to distinguish emitters, Magnify-SOFI can achieve ∼13 nm lateral effective resolution when using the same 1.15 NA objective lens. First, we explored new anchoring agents and found methacrolein, a small molecule used in classic fixation protocols[21,22], provided good biomolecule retention. We next improved gel chemistry by incorporating N,N-dimethylacrylamide acid (DMAA), used in the X10 protocol [7]. We combined DMAA with sodium acrylate (SA), acrylamide (AA), and the crosslinker N,N′-methylenebisacrylamide (Bis) and discovered a mechanically sturdy hydrogel formula composed of 4% (w/v) DMAA, 34% (w/v) SA, 10% (w/v) AA, and 0.01% (w/v) Bis, capable of expanding freshly-preserved mouse brain slices up to 11-fold (Fig. 2a) and FFPE human kidney tissue sections by >8.5-fold (Fig. 2b) in water and after heat denaturation. Low distortion was confirmed in FFPE human kidney and prostate samples when comparing pre-expansion SOFI images with post-expansion Magnify images (Fig. 2c) and we found broad compatibility across tissue types (Fig. 2d). To determine the ground-truth resolution capabilities of Magnify, we first examined the ultrastructure of antibody labeled tubulin in expanded samples. Analysis of the ultrastructure of microtubules in U2OS cells and cilia in human lung organoids (Fig. 4) showed average peak-to-peak distances of 22.68 nm ± 0.71 nm (mean ± s.e.m.) and 24.72 nm ± 0.72 nm (mean ± s.e.m.), similar previous measurements of tubulin width using ExM combined with super resolution imaging techniques [23]. In conclusion, Magnify, a powerful new ExM framework, can preserve an array of biomolecule classes and enables volumetric nanoscale imaging of biological specimens. Magnify is capable of achieving ∼25-nm effective resolution using an ∼280-nm diffraction-limited objective lens on a conventional optical microscope. This effective resolution can be further increased to ∼15 nm if combined with computational methods. Thanks to its broad compatibility across fixation methods used in biomedical research, Magnify can provide effective resolutions comparable to super resolution techniques without the need for expensive, specialized equipment. Additionally, because Magnify is a chemical strategy that does not rely on a specific optical imaging modality, it can be adapted for use with established super resolution optical imaging techniques to achieve higher resolution, or combined with nanoscale imaging modalities such as stimulated Raman scattering [24].
Magnify is a universal molecular anchoring strategy for expansion microscopy
Nature Biotechnology
Expansion microscopy enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a crosslinked water-swellable hydrogel. Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. We describe a strategy called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11 times and facilitates imaging of cells and tissues with effectively around 25-nm resolution using a diffraction-limited objective lens of about 280 nm on conventional optical microscopes or with around 15 nm effective resolution if combined with super-resolution optical fluctuation imaging. We demonstrate Magnify on a broad range of biological specimens, providing insight into nanoscopic subcellular structures, including ...
Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy
Nature biotechnology, 2017
Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity comp...
Visualizing subcellular structures in neuronal tissue with expansion microscopy
2020
ABSTRACTProtein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduces fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. Here we describe a proExM workflow optimized for resolving subcellular organelles (mitochondria and the Golgi apparatus) and reporter-labeled spines in fixed mouse brain tissue. By directly comparing proExM staining and digestion protocols, we found that immunostaining before proExM and using a proteinase K based digestion for 8 hours consistently resulted in the best fluorescence signal to resolve subcellular organelles while maint...
Imaging cellular ultrastructures using expansion microscopy (U-ExM)
Nature Methods
Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy. Cells comprise organelles, large macromolecular assemblies displaying specific structures that for decades could be visualized only by electron microscopy 1. Although super-resolution fluorescence microscopy has evolved as a very powerful method for subdiffractionresolution fluorescence imaging of cells, the visualization of ultrastructural details of macromolecular assemblies remains challenging 2. Recently an innovative method called expansion microscopy (ExM) emerged in which immunolabeled samples are physically expanded, and thus can undergo super-resolution imaging by standard fluorescence microscopy 3,4 (Supplementary Fig. 1a). Alternative ExM protocols such as protein-retention ExM 5 and magnified analysis of the proteome (MAP) 6 have been developed that cross-link proteins in the polymer matrix and allow for postexpansion immunostaining (Supplementary Fig. 1a). However, it remains unclear whether these methods preserve the molecular architecture of organelles. Here we first set out to characterize the macromolecularexpansion performance of established ExM and MAP protocols 4,6. As reference structures, we used isolated Chlamydomonas centrioles, which have a characteristic ninefold microtubule tripletbased symmetry, forming a polarized cylinder ~500 nm long and ~220 nm wide 7 (Supplementary Fig. 1b). We immunolabeled isolated centrioles for α-tubulin, to visualize the centriolar microtubule wall, and for polyglutamylated tubulin (PolyE) present only on the central region of the centriole 7,8. Although the cylindrical nature of the centriole was visible with the PolyE signal in confocal microscopy, it was impossible to visualize the canonical ninefold symmetry of the microtubule triplets (Fig. 1a). Moreover, we noticed antibody competition when we costained for both α-tubulin and PolyE, with both antibodies recognizing epitopes on the C-terminal moiety of tubulin.
ACS Omega, 2020
Four years after its first report, expansion microscopy (ExM) is now being routinely applied in laboratories worldwide to achieve super-resolution imaging on conventional fluorescence microscopes. By chemically anchoring all molecules of interest to the polymer meshwork of an expandable hydrogel, their physical distance is increased by a factor of ∼4−5× upon dialysis in water, resulting in an imprint of the original sample with a lateral resolution up to 50−70 nm. To ensure a correct representation of the original spatial distribution of the molecules, it is crucial to confirm that the expansion is isotropic, preferentially in all three dimensions. To address this, we present an approach to evaluate the local expansion factor within a biological sample and in all three dimensions. We use photobleaching to introduce well-defined threedimensional (3D) features in the cell and, by comparing the size and shape pre-and postexpansion, these features can be used as an intrinsic ruler. In addition, our method is capable of pointing out sample distortions and can be used as a quality control tool for expansion microscopy experiments in biological samples.
Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)
2018
ABSTRACTFor decades, electron microscopy (EM) was the only method able to reveal the ultrastructure of cellular organelles and molecular complexes because of the diffraction limit of optical microscopy. In recent past, the emergence of superresolution fluorescence microscopy enabled the visualization of cellular structures with so far unmatched spatial resolution approaching virtually molecular dimensions. Despite these technological advances, currently super-resolution microscopy does not permit the same resolution level as provided by electron microscopy, impeding the attribution of a protein to an ultrastructural element. Here, we report a novel method of near-native expansion microscopy (UltraExM), enabling the visualization of preserved ultrastructures of macromolecular assemblies with subdiffraction-resolution by standard optical microscopy. UltraExM revealed for the first time the ultrastructural localization of tubulin glutamylation in centrioles. Combined with super-resolut...
Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density
Biophysical Journal, 2010
Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.
Nature Methods, 2022
Cryofixation has proven to be the gold standard for efficient preservation of native cell ultrastructure compared to chemical fixation, but this approach is not widely used in fluorescence microscopy owing to implementation challenges. Here, we develop Cryo-ExM, a method that preserves native cellular organization by coupling cryofixation with expansion microscopy. This method bypasses artifacts associated with chemical fixation and its simplicity will contribute to its widespread use in super-resolution microscopy.
Super-resolution Microscopy Approaches for Live Cell Imaging
Biophysical Journal, 2014
By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.