Expansion microscopy: development and neuroscience applications

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Highlights

  • Classical super-resolution methods are difficult to use across neural circuits.

  • Expansion microscopy physically magnifies specimens to reveal nanoscale features.

  • Expansion microscopy enables visualization of proteins, RNA, and other biomolecules.

  • Expansion microscopy is easy to use, and rapidly spreading throughout the field.

Many neuroscience questions center around understanding how the molecules and wiring in neural circuits mechanistically yield behavioral functions, or go awry in disease states. However, mapping the molecules and wiring of neurons across the large scales of neural circuits has posed a great challenge. We recently developed expansion microscopy (ExM), a process in which we physically magnify biological specimens such as brain circuits. We synthesize throughout preserved brain specimens a dense, even mesh of a swellable polymer such as sodium polyacrylate, anchoring key biomolecules such as proteins and nucleic acids to the polymer. After mechanical homogenization of the specimen-polymer composite, we add water, and the polymer swells, pulling biomolecules apart. Due to the larger separation between molecules, ordinary microscopes can then perform nanoscale resolution imaging. We here review the ExM technology as well as applications to the mapping of synapses, cells, and circuits, including deployment in species such as Drosophila, mouse, non-human primate, and human.

Introduction

At the core of many neuroscience questions, ranging from understanding how memories are encoded, to how neurons transform sensory inputs into motor outputs, to how emotions and decisions are implemented, is a need to understand how molecules and cells in neural circuits are organized to yield complex emergent functions. Understanding the nature of brain disorders, and pointing the way to new therapeutics, is also increasingly demanding a knowledge of how brain cells, molecular cascades, and connections change in disease states. Ideally one would be able to map biomolecules such as neurotransmitters, receptors, and ion channels, across the spatial extents of neurons and neural circuits. Traditional microscopes are limited by diffraction, and thus specialized technologies have been required to perform imaging with nanoscale precision. Electron microscopy is capable of nanoscale resolution, and has yielded many insights into the wiring diagrams of neural circuits [1, 2], but typically yields little molecular information about the molecules in those circuits. Super-resolution light microscopy methods have powerfully revealed many molecular features of neurons at the nanoscale level [3, 4], but such methods are difficult to apply to extended 3-D specimens, such as neural circuits, due to their speed and complexity. To address the need for a method of imaging extended 3-D objects such as neural circuits, with molecular information, at nanoscale resolution, we recently developed a novel modality of imaging. In contrast to earlier methods of nanoscale imaging that magnify information emitted from a specimen, we physically magnify the specimen itself [5••].

In this new methodology, which we call expansion microscopy (ExM, schematized in Figure 1a–e), we synthesize a dense, interconnected web of a swellable polymer, such as sodium polyacrylate, throughout a preserved specimen such as a brain specimen. The polymer is very dense, such that the distance between adjacent polymer threads is on the order of the dimension of a biomolecule. We anchor biomolecules such as proteins or RNA, or labels bound to those biomolecules (such as antibodies), to the polymer network via covalently binding anchoring molecules. We treat the specimen with heat, detergent, and/or enzymes to mechanically homogenize the specimen so that it can expand evenly, and then finally we add water. The swellable polymer absorbs the water, and expands, bringing the anchored biomolecules or labels along (Figure 1f). The net result is that biomolecules or labels that are initially localized within the diffraction limit of a traditional microscope, are now separated in space to distances far enough that they can now be resolved. The specimen also becomes completely transparent, having become mostly water (Figure 1g).

ExM builds from two sets of ideas that go back into the late 1970s and early 1980s. Around that time, the physicist Toyoichi Tanaka at MIT was creating and studying the physics of swellable gels [6], and found that they could swell many orders of magnitude in volume in ways that could be precisely described via phase transition mathematics. Around the same time, Peter Hausen and Christine Dreyer at the Max Planck Institute developed polymer hydrogel embedding of fixed tissues for the enhancement of imaging, synthesizing polyacrylamide networks throughout preserved specimens [7]. ExM fuses these two old concepts to enable physical magnification of specimens, with precision down to the nanoscale.

In this review, we first discuss the principles of how ExM works, discussing some of the rapidly exploding family of protocols that have been invented in the past few years that are making ExM easier to use and more powerful, and then we discuss some current applications in the field of neuroscience.

Section snippets

Principles of how expansion microscopy works

Since our discovery of expansion microscopy, accompanied by a proof-of-concept protocol and validation data showing its high performance in cultured mammalian cells and mouse brain tissue, published in 2015 [5••], we have developed several variants specialized for simple visualization of proteins [8] and RNA [9••] using off-the-shelf chemicals, variants that can expand cells and tissues to much greater extents than the original protocol [10••], and variants that can easily be applied to human

Applications of ExM to neuroscience

From our earliest paper on ExM, we showed that ExM could be used to visualize synaptic contacts between neurons in brain circuits, for example, in the mouse hippocampus (Figure 2a–d and Ref. [5••]). In particular, with ExM [5••], proExM [8], or iExM [10••], one can visualize synapses and synaptic proteins (e.g. excitatory and inhibitory neurotransmitter receptors, presynaptic scaffolding proteins, postsynaptic scaffolding proteins, neurotransmitter synthesis enzymes, etc.) in the context of

Common problems and strategies to overcome them

As with any new technology, early adopters will need to confront potential problems in order to deploy expansion microscopy into their scientific field. This also presents opportunities for refinement and innovation, as various groups have published papers applying or validating expansion microscopy in new contexts like Drosophila [18, 32], zebrafish [26], or human brain [21]. One common problem that people encounter early in their experiences with expansion microscopy samples regards the

Acknowledgements

For funding E.S.B. acknowledges the HHMI-Simons Fellowship, John Doerr, the Open Philanthropy project, IARPA D16PC00008, NIH Grants 1R01MH103910, 1RM1HG008525, 1R01MH110932, 1R01EB024261and 1R01NS102727, the Cancer Research UK Grand Challenge, and U. S. Army Research Laboratory and the U. S. Army Research Office under contract/Grant Number W911NF1510548. We thank all members of the Synthetic Neurobiology group for helpful discussions.

Conflict of interest statement

ESB is co-founder of a company commercializing clinical

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