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.

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.

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.

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.

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.

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.

Expansion microscopy of .

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 is challenged by its cuticle, which is stiff and impermeable to antibodies.

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.