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Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultra-structural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.

© 2017 Nature America, Inc. All rights reserved. We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Lithographic nanofabrication is often limited to successive fabrication of two-dimensional (2D) layers. We present a strategy for the direct assembly of 3D nanomaterials consisting of metals, semiconductors, and biomolecules arranged in virtually any 3D geometry. We used hydrogels as scaffolds for volumetric deposition of materials at defined points in space. We then optically patterned these scaffolds in three dimensions, attached one or more functional materials, and then shrank and dehydrated them in a controlled way to achieve nanoscale feature sizes in a solid substrate. We demonstrate that our process, Implosion Fabrication (ImpFab), can directly write highly conductive, 3D silver nanostructures within an acrylic scaffold via volumetric silver deposition. Using ImpFab, we achieve resolutions in the tens of nanometers and complex, non–self-supporting 3D geometries of interest for optical metamaterials. ©2017, ONR (no. N00014-17-1-2977), NIH (no. 1R01EB024261), NIH (no. 1U01MH106011), NIH Director’s Pioneer Award (no. 1DP1NS087724), NIH (no. 1RM1HG008525), NIH (no. 1R24MH106075), National Science Foundation Graduate Research Fellowship Program (award no. 1122374)

Expansion microscopy (ExM) is a recently developed technique that enables nanoscale-resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes via isotropic physical expansion of the specimens before imaging. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by ∼4.5 fold linearly when immersed in water. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged. Here we describe best-practice, step-by-step ExM protocols for performing analysis of proteins (protein retention ExM, or proExM) as well as RNAs (expansion fluorescence in situ hybridization, or ExFISH), using chemicals and hardware found in a typical biology lab. Furthermore, a detailed protocol for handling and mounting expanded samples and for imaging them with confocal and light-sheet microscopes is provided., NIH (Grant 1R01NS102727), NIH (Grant 1R01EB024261), NIH (Grant 1DP1NS087724), U.S. Army Research Office (Grant W911NF1510548), U.S. Army Research Office (Grant 1RM1HG008525), IARPA (Grant D16PC00008)

The dopamine systems of the brain powerfully influence movement and motivation. We demonstrate that striatonigral fibers originating in striosomes form highly unusual bouquet-like arborizations that target bundles of ventrally extending dopamine-containing dendrites and clusters of their parent nigral cell bodies. Retrograde tracing showed that these clustered cell bodies in turn project to the striatum as part of the classic nigrostriatal pathway. Thus, these striosome-dendron formations, here termed "striosome-dendron bouquets," likely represent subsystems with the nigro-striato-nigral loop that are affected in human disorders including Parkinson's disease. Within the bouquets, expansion microscopy resolved many individual striosomal fibers tightly intertwined with the dopamine-containing dendrites and also with afferents labeled by glutamatergic, GABAergic, and cholinergic markers and markers for astrocytic cells and fibers and connexin 43 puncta. We suggest that the striosome-dendron bouquets form specialized integrative units within the dopamine-containing nigral system. Given evidence that striosomes receive input from cortical regions related to the control of mood and motivation and that they link functionally to reinforcement and decision-making, the striosome-dendron bouquets could be critical to dopamine-related function in health and disease.

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such “hybrid microscopy” methods—combining physical and optical magnifications—can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes (“mini-microscopes”), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics—a process we refer to as Expansion Mini-Microscopy (ExMM)—is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places., National Institutes of Health (U.S.) (EB012597), National Institutes of Health (U.S.) (AR057837), National Institutes of Health (U.S.) (DE021468), National Institutes of Health (U.S.) (HL099073), National Institutes of Health (U.S.) (R56AI105024), United States. Office of Naval Research. Young Investigator Program, Presidential Early Career Award for Scientists and Engineers, MIT International Science and Technology Initiatives, Massachusetts Institute of Technology. Media Laboratory, National Institutes of Health (U.S.) (Transformative Award NIH 1R01MH103910), National Institutes of Health (U.S.) (Transformative Award NIH 1U01MH106011), National Institutes of Health (U.S.) (Director’s Pioneer Award 1DP1NS087724), New York Stem Cell Foundation (New York Stem Cell Foundation-Robertson Award), Simons Foundation

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes., United States. National Institutes of Health (1R01GM104948), United States. National Institutes of Health (1DP1NS087724), United States. National Institutes of Health ( NIH 1R01EY023173), United States. National Institutes of Health (1U01MH106011)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016., Cataloged from PDF version of thesis., Includes bibliographical references (pages 70-76)., Until the past decade, optical microscopy of biological specimens was strongly limited by diffraction and scattering, affecting imaging resolution and depth, respectively. Now, numerous methods are available to overcome each of these limitations, but sub-diffraction limited resolution imaging over large volumes of scattering tissue is still a challenge. This work concerns the development of a new method, Expansion Microscopy (ExM) for achieving effect sub-diffraction-limited optical images in biological specimens. In ExM, the specimen is embedded in a swellable gel material to which fluorescent probes are chemically anchored. The embedded tissue is strongly digested so that it will not hinder uniform expansion driven by the gel. The gel with embedded, fragmented tissue is washed in water, triggering expansion of around 4-fold in each dimension. A variant of the method, ExM with Protein Retention (proExM) is presented that allows proteins themselves, rather than fluorescent probes, to be anchored by a small molecule cross-linker to the gel, so that the method may be carried out entirely with commercial components and standard antibodies., by Paul W. Tillberg., Ph. D.

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles., National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127), National Institutes of Health (U.S.) (Director's Innovator Award DP2 OD002989), David & Lucile Packard Foundation (Award in Science and Engineering), Sanofi Aventis (Firm), Foxconn International Holdings Ltd., Hertz Foundation (Fellowship), University Grants Committee (Hong Kong, China) (Early Career Award 125012), National Natural Science Foundation (China) (81201164), ITC (ITS/376/13), Chinese University of Hong Kong (Grant 9610215), Chinese University of Hong Kong (Grant 7200269)

Available in PMC 2015 July 30., In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~107 cubic micrometers of the mouse hippocampus with a conventional confocal microscope., National Institutes of Health (U.S.) (NIH Director’s Pioneer Award 1DP1NS087724), National Institutes of Health (U.S.) (NIH Transformative Research Award 1R01MH103910-01), New York Stem Cell Foundation (Robertson Investigator Award), National Science Foundation (U.S.). Center for Brains, Minds and Machines (CBMM) (NSF CCF-1231216), National Science Foundation (U.S.) (NSF CAREER Award CBET 1053233)

Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin’s local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology., National Institutes of Health (U.S.) (grant NIH 1DP2OD002002), National Institutes of Health (U.S.) (grant NIH 1R01DA029639), National Institutes of Health (U.S.) (grant NIH 1R01NS075421), National Institutes of Health (U.S.) (grant NIH 1RC1MH088182), National Science Foundation (U.S.) (NSF CAREER Award CBET 1053233), United States. Defense Advanced Research Projects Agency (DARPA Living Foundries, Contract HR0011-12-C-0068), New York Stem Cell Foundation (Robertson Investigator Award), Damon Runyon Cancer Research Foundation (DRG 2095-11), Fannie and John Hertz Foundation, National Science Foundation (U.S.) (Graduate Research Fellowship under grant no. 1122374), Massachusetts Institute of Technology. Synthetic Intelligence Laboratory (project)

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2013., Cataloged from PDF version of thesis., Includes bibliographical references (p. 30-31)., The aim of this work is to increase the multiplexing capabilities of electron and super resolution optical microscopy. This will be done through the development of molecular-scale barcodes that can be resolved in one of the two high resolution imaging modes. In the optical domain, the number of colors available in stochastic optical reconstruction microscopy (STORM) will be increased by taking advantage of not only the spectral differences between STORM fluorophores but their kinetic properties as well. In the electron microscopy domain, the recently developed electron contrast-generating protein miniSOG will be concatenated to produce fully genetically encoded barcodes that can be resolved using standard transmission electron microscopy techniques. At the time of writing, the hardware for a STORM microscope has been assembled. Single molecule fluorescence blinking has been observed, though the imaging buffer still needs to be optimized for imaging. Concatamers of miniSOG have been generated and can be expressed in HEK cells and photo-oxidized., by Paul W. Tillberg., S.M.