Diffraction-limited Microscopy Research Articles

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

Visualization of the membrane surface and cytoskeleton of oligodendrocyte progenitor cell growth cones using a combination of scanning ion conductance and four times expansion microscopy.

Growth cones of oligodendrocyte progenitor cells (OPCs) are challenging to investigate with conventional light microscopy due to their small size. Especially substructures such as filopodia, lamellipodia and their underlying cytoskeleton are difficult to resolve with diffraction limited microscopy. Light microscopy techniques, which surpass the diffraction limit such as stimulated emission depletion microscopy, often require expensive setups and specially trained personnel rendering them inaccessible to smaller research groups. Lately, the invention of expansion microscopy (ExM) has enabled super-resolution imaging with any light microscope without the need for additional equipment. Apart from the necessary resolution, investigating OPC growth cones comes with another challenge: Imaging the topography of membranes, especially label- and contact-free, is only possible with very few microscopy techniques one of them being scanning ion conductance microscopy (SICM). We here present a new imaging workflow combining SICM and ExM, which enables the visualization of OPC growth cone nanostructures. We correlated SICM recordings and ExM images of OPC growth cones captured with a conventional widefield microscope. This enabled the visualization of the growth cones' membrane topography as well as their underlying actin and tubulin cytoskeleton.

Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

Over the last decade, single-molecule localization microscopy (SMLM) has developed into a set of powerful techniques that have improved spatial resolution over diffraction-limited microscopy and demonstrated the ability to resolve biological features down to a few tens of nanometers. We introduce a single molecule-based scanning SMLM (scanSMLM) system that enables rapid volume imaging. Along with epi-illumination, the system employs a scanning-based 4f detection for volume imaging. The 4f system comprises a combination of an electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of an electrically-tunable lens (ETL) in a 4f detection system, the selectivity of the axial object plane is achieved, for which the image forms in the image/detector plane. So, in principle, one can scan the object volume by just altering the aperture of ETL. Two schemes were adopted to carry out volume imaging: cyclic scan and conventional scan. The cyclic scheme scans the volume in each scan cycle, whereas plane-wise scanning is performed in the conventional scheme. Hence, the cyclic scan ensures uniform dwell time on each frame during data collection, thereby evenly distributing photobleaching throughout the cell volume. With a minimal change in the system hardware (requiring the addition of an ETL lens and related electronics for step-voltage generation) in the existing SMLM system, volume scanning (along the z-axis) can be achieved. To calibrate and derive critical system parameters, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, scanSMLM is employed to visualize the architecture of actin-filaments and the distribution of Meos-Tom20 molecules on the mitochondrial membrane. The technique is further exploited to understand the clustering of Hemagglutinin (HA) protein single molecules in a transfected cell for studying Influenza-A disease progression. The system, for the first time, enabled 3D visualization of HA distribution that revealed HA cluster formation spanning the entire cell volume, post 24 hrs of transfection. Critical biophysical parameters related to HA clusters (density, the number of HA molecules per cluster, axial span, fraction of clustered molecules, and others) are also determined, giving an unprecedented insight into Influenza-A disease progression at the single-molecule level.

Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates.

The intracellular liquid-liquid phase separation (LLPS) of biomolecules gives rise to condensates that act as membrane-less organelles with vital functions. FUS, an RNA-binding protein, natively forms condensates through LLPS and further provides a model system for the often disease-linked liquid-to-solid transition of biomolecular condensates during aging. However, the mechanism of such maturation processes, as well as the structural and physical properties of the system, remains unclear, partly attributable to difficulties in resolving the internal structures of the micrometer-sized condensates with diffraction-limited optical microscopy. Harnessing a set of multidimensional super-resolution microscopy tools that uniquely map out local physicochemical parameters through single-molecule spectroscopy, here, we uncover nanoscale heterogeneities in FUS condensates and elucidate their evolution over aging. Through spectrally resolved single-molecule localization microscopy (SR-SMLM) with a solvatochromic dye, we unveil distinct hydrophobic nanodomains at the condensate surface. Through SMLM with a fluorogenic amyloid probe, we identify these nanodomains as amyloid aggregates. Through single-molecule displacement/diffusivity mapping (SMdM), we show that such nanoaggregates drastically impede local diffusion. Notably, upon aging or mechanical shears, these nanoaggregates progressively expand on the condensate surface, thus leading to a growing low-diffusivity shell while leaving the condensate interior diffusion-permitting. Together, beyond uncovering fascinating structural arrangements and aging mechanisms in the single-component FUS condensates, the demonstrated synergy of multidimensional super-resolution approaches in this study opens new paths for understanding LLPS systems at the nanoscale.

Early detection of lung cancer using artificial intelligence-enhanced optical nanosensing of chromatin alterations in field carcinogenesis

Supranucleosomal chromatin structure, including chromatin domain conformation, is involved in the regulation of gene expression and its dysregulation has been associated with carcinogenesis. Prior studies have shown that cells in the buccal mucosa carry a molecular signature of lung cancer among the cigarette-smoking population, the phenomenon known as field carcinogenesis or field of injury. Thus, we hypothesized that chromatin structural changes in buccal mucosa can be predictive of lung cancer. However, the small size of the chromatin chain (approximately 20 nm) folded into chromatin packing domains, themselves typically below 300 nm in diameter, preclude the detection of alterations in intradomain chromatin conformation using diffraction-limited optical microscopy. In this study, we developed an optical spectroscopic statistical nanosensing technique to detect chromatin packing domain changes in buccal mucosa as a lung cancer biomarker: chromatin-sensitive partial wave spectroscopic microscopy (csPWS). Artificial intelligence (AI) was applied to csPWS measurements of chromatin alterations to enhance diagnostic performance. Our AI-enhanced buccal csPWS nanocytology of 179 patients at two clinical sites distinguished Stage-I lung cancer versus cancer-free controls with an area under the ROC curve (AUC) of 0.92 ± 0.06 for Site 1 (in-state location) and 0.82 ± 0.11 for Site 2 (out-of-state location).

Presynapses contain distinct actin nanostructures.

The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding of its roles in synaptic physiology. In this work, we measure and visualize presynaptic actin by diffraction-limited and super-resolution microscopy, thanks to a validated model of bead-induced presynapses in cultured neurons. We identify a major population of actin-enriched presynapses that concentrates more presynaptic components and shows higher synaptic vesicle cycling than their non-enriched counterparts. Pharmacological perturbations point to an optimal actin amount and the presence of distinct actin structures within presynapses. We directly visualize these nanostructures using Single Molecule Localization Microscopy (SMLM), defining three distinct types: an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. Finally, CRISPR-tagging of endogenous actin allows us to validate our results in natural synapses between cultured neurons, confirming the role of actin enrichment and the presence of three types of presynaptic actin nanostructures.

Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Dimerization of iLID optogenetic proteins observed using 3D single-molecule tracking in live E. coli

3D single-molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and cellular environments. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments. When investigating cellular processes, real-time perturbations to protein spatial distributions is preferable to permanent genetic deletion of an essential protein. For example, optogenetic dimerization systems can be used to manipulate protein spatial distributions that could offer a means to deplete specific diffusive states observed in single-molecule tracking experiments. Here, we evaluate the performance of the iLID optogenetic system in living E. coli cells using diffraction-limited microscopy and 3D single-molecule tracking. We observed a robust optogenetic response in protein spatial distributions after 488 nm laser activation. Surprisingly, 3D single-molecule tracking results indicate activation of the optogenetic response when illuminating with high-intensity light with wavelengths at which there is minimal photon absorbance by the LOV2 domain. The preactivation can be minimized through the use of iLID system mutants, and titration of protein expression levels.

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscopefor reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

A computational suite for the structural and functional characterization of amyloid aggregates

We developed the aggregate characterization toolkit (ACT), a fully automated computational suite based on existing and widely used core algorithms to measure the number, size, and permeabilizing activity of recombinant and human-derived aggregates imaged with diffraction-limited and super-resolution microscopy methods at high throughput. We have validated ACT on simulated ground-truth images of aggregates mimicking those from diffraction-limited and super-resolution microscopies and showcased its use in characterizing protein aggregates from Alzheimer's disease. ACT is developed for high-throughput batch processing of images collected from multiple samples and is available as an open-source code. Given its accuracy, speed, and accessibility, ACT is expected to be a fundamental tool in studying human and non-human amyloid intermediates, developing early disease stage diagnostics, and screening for antibodies that bind toxic and heterogeneous human amyloid aggregates.

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

Dendritic spinule-mediated structural synaptic plasticity: Implications for development, aging, and psychiatric disease.

Dendritic spines are highly dynamic and changes in their density, size, and shape underlie structural synaptic plasticity in cognition and memory. Fine membranous protrusions of spines, termed dendritic spinules, can contact neighboring neurons or glial cells and are positively regulated by neuronal activity. Spinules are thinner than filopodia, variable in length, and often emerge from large mushroom spines. Due to their nanoscale, spinules have frequently been overlooked in diffraction-limited microscopy datasets. Until recently, our knowledge of spinules has been interpreted largely from single snapshots in time captured by electron microscopy. We summarize herein the current knowledge about the molecular mechanisms of spinule formation. Additionally, we discuss possible spinule functions in structural synaptic plasticity in the context of development, adulthood, aging, and psychiatric disorders. The literature collectively implicates spinules as a mode of structural synaptic plasticity and suggests the existence of morphologically and functionally distinct spinule subsets. A recent time-lapse, enhanced resolution imaging study demonstrated that the majority of spinules are small, short-lived, and dynamic, potentially exploring their environment or mediating retrograde signaling and membrane remodeling via trans-endocytosis. A subset of activity-enhanced, elongated, long-lived spinules is associated with complex PSDs, and preferentially contacts adjacent axonal boutons not presynaptic to the spine head. Hence, long-lived spinules can form secondary synapses with the potential to alter synaptic connectivity. Published studies further suggest that decreased spinules are associated with impaired synaptic plasticity and intellectual disability, while increased spinules are linked to hyperexcitability and neurodegenerative diseases. In summary, the literature indicates that spinules mediate structural synaptic plasticity and perturbations in spinules can contribute to synaptic dysfunction and psychiatric disease. Additional studies would be beneficial to further delineate the molecular mechanisms of spinule formation and determine the exact role of spinules in development, adulthood, aging, and psychiatric disorders.

Expansion Microscopy of Plant Cells (PlantExM).

Expansion microscopy (ExM) achieves super-resolution imaging without the need for sophisticated super-resolution microscopy hardware through a combination of physical and optical magnification. Samples are fixed, stained, and embedded in a swellable gel. Following cross-linking of fluorophores to the gel matrix, the components of the sample are digested away and the gel expanded in water. Labeled objects which are too close to be resolved by diffraction-limited microscopy are moved far enough apart that these can now be resolved as individual objects on a standard confocal. Originally developed for animal cells and tissues, ExM for plants requires the additional consideration of cell wall digestion. Super-resolution can be limited in plants due to the size of cells, light scattering of tissues, and variations in refractive index. By removing the components which cause these limitations, ExM opens up the possibility of super-resolution at depth within plant tissues for the first time. Here we describe our method for PlantExM which is optimized for cytoskeleton resolution, which, when also coupled with compatible optical super-resolution technologies, can produce images of the plant cytoskeleton in unprecedented detail.

In situ silver nanoparticle development for molecular-specific biological imaging via highly accessible microscopies.

In biological studies and diagnoses, brightfield (BF), fluorescence, and electron microscopy (EM) are used to image biomolecules inside cells. When compared, their relative advantages and disadvantages are obvious. BF microscopy is the most accessible of the three, but its resolution is limited to a few microns. EM provides a nanoscale resolution, but sample preparation is time-consuming. In this study, we present a new imaging technique, which we termed decoration microscopy (DecoM), and quantitative investigations to address the aforementioned issues in EM and BF microscopy. For molecular-specific EM imaging, DecoM labels proteins inside cells using antibodies bearing 1.4 nm gold nanoparticles (AuNPs) and grows silver layers on the AuNPs' surfaces. The cells are then dried without buffer exchange and imaged using scanning electron microscopy (SEM). Structures labeled with silver-grown AuNPs are clearly visible on SEM, even they are covered with lipid membranes. Using stochastic optical reconstruction microscopy, we show that the drying process causes negligible distortion of structures and that less structural deformation could be achieved through simple buffer exchange to hexamethyldisilazane. Using DecoM, we visualize the nanoscale alterations in microtubules by microtubule-severing proteins that cannot be observed with diffraction-limited fluorescence microscopy. We then combine DecoM with expansion microscopy to enable sub-micron resolution BF microscopy imaging. We first show that silver-grown AuNPs strongly absorb white light, and the structures labeled with them are clearly visible on BF microscopy. We then show that the application of AuNPs and silver development must follow expansion to visualize the labeled proteins clearly with sub-micron resolution.

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

ABSTRACT Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images. Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

Protein misfolding in the form of fibrils or spherulites is involved in a spectrum of pathological abnormalities. Our current understanding of protein aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction-limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies of human insulin, below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of microscopically identical aggregates, e.g., amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

Dynamic Heterochromatin States in Anisotropic Nuclei of Cells on Aligned Nanofibers.

The cancer cell nucleus deforms as it invades the interstitial spaces in tissues and the tumor microenvironment. While alteration of the chromatin structure in a deformed nucleus is expected and documented, the chromatin structure in the nuclei of cells on aligned matrices has not been elucidated. In this work we elucidate the spatiotemporal organization of heterochromatin in the elongated nuclei of cells on aligned nanofibers with stimulated emission depletion nanoscopy and fluorescence correlation spectroscopy. We show that the anisotropy of nuclei is sufficient to drive H3K9me3-heterochromatin alterations, with enhanced H3K9me3 nanocluster compaction and aggregation states that otherwise are indistinguishable from diffraction-limited microscopy. We interrogated the higher-order heterochromatin structures within major chromatin compartments in anisotropic nuclei and discovered a wider spatial dispersion of nanodomain clusters in the nucleoplasm and condensed larger nanoclusters near the periphery and pericentromeric heterochromatin. Upon examining the spatiotemporal dynamics of heterochromatin in anisotropic nuclei, we observed reduced mobility of the constitutive heterochromatin mark H3K9me3 and the associated heterochromatin protein 1 (HP1α) at the nucleoplasm and periphery regions, correlating with increased viscosity and changes in gene expression. Since heterochromatin remodeling is crucial to genome integrity, our results reveal an unconventional H3K9me3 heterochromatin distribution, providing cues to an altered chromatin state due to perturbations of the nuclei in aligned fiber configurations.

PYME: an integrated platform for high-throughput nanoscopy

Single-molecule localization microscopy (SMLM) offers a ∼10-fold improvement in resolution over conventional, diffraction-limited fluorescence microscopy, which comes at the expense of acquisition time, data volume, and analysis overhead. For (d)STORM/(F)PALM, raw series typically consist of 10,000 to 100,000 frames acquired at 50 frames per second (FPS), meaning a day of manual imaging yields only on the order of ten fields of view. While efforts have been made to ease the tedium of acquisition by automating SMLM microscopes, the achieved throughputs are not dissimilar from manual imaging.

Non-telecentric two-photon microscopy for 3D random access mesoscale imaging

Diffraction-limited two-photon microscopy permits minimally invasive optical monitoring of neuronal activity. However, most conventional two-photon microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric optical design, we present a low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.

Epitope-preserving magnified analysis of proteome (eMAP).

Synthetic tissue-hydrogel methods have enabled superresolution investigation of biological systems using diffraction-limited microscopy. However, chemical modification by fixatives can cause loss of antigenicity, limiting molecular interrogation of the tissue gel. Here, we present epitope-preserving magnified analysis of proteome (eMAP) that uses purely physical tissue-gel hybridization to minimize the loss of antigenicity while allowing permanent anchoring of biomolecules. We achieved success rates of 96% and 94% with synaptic antibodies for mouse and marmoset brains, respectively. Maximal preservation of antigenicity allows imaging of nanoscopic architectures in 1000-fold expanded tissues without additional signal amplification. eMAP-processed tissue gel can endure repeated staining and destaining without epitope loss or structural damage, enabling highly multiplexed proteomic analysis. We demonstrated the utility of eMAP as a nanoscopic proteomic interrogation tool by investigating molecular heterogeneity in inhibitory synapses in the mouse brain neocortex and characterizing the spatial distributions of synaptic proteins within synapses in mouse and marmoset brains.