Q&A: Expansion microscopy

Expansion microscopy (ExM) is a recently invented technology that uses swellable charged polymers, synthesized densely and with appropriate topology throughout a preserved biological specimen, to physically magnify the specimen 100-fold in volume, or more, in an isotropic fashion. ExM enables nanoscale resolution imaging of preserved samples on inexpensive, fast, conventional microscopes. How does ExM work? How good is its performance? How do you get going on using it? In this Q & A, we provide the answers to these and other questions about this new and rapidly spreading toolbox

Expansion microscopy of zebrafish for neuroscience and developmental biology studies

Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.National Institutes of Health (U.S.) (Grant 1R01EB024261)National Institutes of Health (U.S.) (Grant 1R01MH110932)National Institutes of Health (U.S.) (Grant 2R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS087950)National Institutes of Health (U.S.) (Grant 1U01MH106011

Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution

© 2019 American Association for the Advancement of Science. All Rights Reserved. Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast

Expansion Microscopy for Beginners: Visualizing Microtubules in Expanded Cultured HeLa Cells

© 2020 The Authors. Expansion microscopy (ExM) is a technique that physically expands preserved cells and tissues before microscope imaging, so that conventional diffraction-limited microscopes can perform nanoscale-resolution imaging. In ExM, biomolecules or their markers are linked to a dense, swellable gel network synthesized throughout a specimen. Mechanical homogenization of the sample (e.g., by protease digestion) and the addition of water enable isotropic swelling of the gel, so that the relative positions of biomolecules are preserved. We previously presented ExM protocols for analyzing proteins and RNAs in cells and tissues. Here we describe a cookbook-style ExM protocol for expanding cultured HeLa cells with immunostained microtubules, aimed to help newcomers familiarize themselves with the experimental setups and skills required to successfully perform ExM. Our aim is to help beginners, or students in a wet-lab classroom setting, learn all the key steps of ExM. © 2020 The Authors

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.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

In situ structural analysis of Golgi intracisternal protein arrays

We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had similar to 6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function

Nanoscale Imaging of RNA with Expansion Microscopy

The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy (ExM) of RNA. We developed a small molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples

Abstract Background Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. Results To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. Conclusions The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes

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

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

© 2020 Elsevier Inc. One-photon fluorescent imaging of calcium signals can capture the activity of hundreds of neurons across large fields of view but suffers from crosstalk from neuropil. Shemesh et al. engineer cell-body-targeted variants of fluorescent calcium indicators and show in mice and zebrafish that artifactual spikes and correlations are greatly reduced