EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Expansion Microscopy: Scalable and Convenient Super-Resolution Microscopy.

Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 µm thick.

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

Iterative expansion microscopy.

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.

Striosome-dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons.

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.

Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues.

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.

Ten-fold Robust Expansion Microscopy.

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.

Multimodal mapping of cell types and projections in the central nucleus of the amygdala.

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).

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 ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

Rapid reconstruction of neural circuits using tissue expansion and light sheet microscopy.

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: Protocols for Imaging Proteins and RNA in Cells and Tissues.

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. © 2018 by John Wiley & Sons, Inc.

3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds.

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.

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.

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.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

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.

Optical imaging. Expansion microscopy.

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 ~10(7) cubic micrometers of the mouse hippocampus with a conventional confocal microscope.

A fully genetically encoded protein architecture for optical control of peptide ligand concentration.

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.