Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

ExCel: Super-Resolution Imaging of C. elegans with Expansion Microscopy.

Studies of C. elegans will benefit from a powerful method for super-resolution imaging of proteins and mRNAs at any 3-D locations throughout the entire animal. Conventional methods of super-resolution imaging in C. elegans, such as STORM, PALM, SR-SIM and STED, are limited by imaging depths that are insufficient to map the entire depth of adult worms, and involve hardware that may not be accessible to all labs. We recently developed expansion of C. elegans (ExCel), a method for physically magnifying fixed whole animals of C. elegans with high isotropy, which provides effective resolutions finer than the diffraction limit, across the entire animal, on conventional confocal microscopes. In this chapter, we present a family of three detailed ExCel protocols. The standard ExCel protocol features simultaneous readout of diverse molecules (fluorescent proteins, RNA, DNA, and general anatomy), all at ~70 nm resolution (~3.5× linear expansion). The epitope-preserving ExCel protocol enables imaging of endogenous proteins with off-the-shelf antibodies, at a ~ 100 nm resolution (~2.8× linear expansion). The iterative ExCel protocol allows readout of fluorescent proteins at ~25 nm resolution (~20× linear expansion). The protocols described here comprise a versatile toolbox for super-resolution imaging of C. elegans.

A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy.

Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging using conventional microscopes. Current ExM methods permeate specimens with free-radical-chain-growth-polymerized polyacrylate hydrogels, whose network structure limits the local isotropy of expansion as well as the preservation of morphology and shape at the nanoscale. Here we report that ExM is possible using hydrogels that have a more homogeneous network structure, assembled via non-radical terminal linking of tetrahedral monomers. As with earlier forms of ExM, such 'tetra-gel'-embedded specimens can be iteratively expanded for greater physical magnification. Iterative tetra-gel expansion of herpes simplex virus type 1 (HSV-1) virions by ~10× in linear dimension results in a median spatial error of 9.2 nm for localizing the viral envelope layer, rather than 14.3 nm from earlier versions of ExM. Moreover, tetra-gel-based expansion better preserves the virion spherical shape. Thus, tetra-gels may support ExM with reduced spatial errors and improved local isotropy, pointing the way towards single-biomolecule accuracy ExM.

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems.

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.

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics.

In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.

Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator.

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.

Expansion microscopy of C. elegans.

We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal.

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.

Multi-well imaging of development and behavior in Caenorhabditis elegans.

BACKGROUND: The nematode Caenorhabditis elegans is widely used as a model for understanding the neuronal and genetic bases of behavior. Recent studies have required longitudinal assessment of individual animal's behavior over extended periods. NEW METHOD: Here we present a technique for automated monitoring of multiple worms for several days. Our method uses an array of plano-concave glass wells containing standard agar media. The concave well geometry allows worms to be imaged even at the edge of the agar surface and prevents them from burrowing under the agar. We transfer one worm or embryo into each well, and perform imaging of the array of wells using a single camera. Machine vision software is used to quantify size, activity, and/or fluorescence of each worm over time. RESULTS: We demonstrate the utility of our method in two applications: (1) quantifying behavioral quiescence and developmental rate in wild-type and mutant animals, and (2) characterizing differences in mating behavior between two C. elegans strains. COMPARISON WITH EXISTING METHOD(S): Current techniques for tracking behavior in identified worms are generally restricted to imaging either single animals or have not been shown to work with arbitrary developmental stages; many are also technically complex. Our system works with up to 24 animals of any stages and is technically simple. CONCLUSIONS: Our multi-well imaging method is a powerful tool for quantification of long-term behavioral phenotypes in C. elegans.