Molecularly defined and spatially resolved cell atlas of the whole mouse brain.

In mammalian brains, millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells have impeded our understanding of the molecular and cellular basis of brain function. Recent advances in spatially resolved single-cell transcriptomics have enabled systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1-3, including several brain regions (for example, refs. 1-11). However, a comprehensive cell atlas of the whole brain is still missing. Here we imaged a panel of more than 1,100 genes in approximately 10 million cells across the entire adult mouse brains using multiplexed error-robust fluorescence in situ hybridization12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating multiplexed error-robust fluorescence in situ hybridization and single-cell RNA sequencing data. Using this approach, we generated a comprehensive cell atlas of more than 5,000 transcriptionally distinct cell clusters, belonging to more than 300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of this atlas to the mouse brain common coordinate framework allowed systematic quantifications of the cell-type composition and organization in individual brain regions. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes of cells. Finally, this high-resolution spatial map of cells, each with a transcriptome-wide expression profile, allowed us to infer cell-type-specific interactions between hundreds of cell-type pairs and predict molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a foundation for functional investigations of neural circuits and their dysfunction in health and disease.

Conservation and divergence of cortical cell organization in human and mouse revealed by MERFISH.

The human cerebral cortex has tremendous cellular diversity. How different cell types are organized in the human cortex and how cellular organization varies across species remain unclear. In this study, we performed spatially resolved single-cell profiling of 4000 genes using multiplexed error-robust fluorescence in situ hybridization (MERFISH), identified more than 100 transcriptionally distinct cell populations, and generated a molecularly defined and spatially resolved cell atlas of the human middle and superior temporal gyrus. We further explored cell-cell interactions arising from soma contact or proximity in a cell type-specific manner. Comparison of the human and mouse cortices showed conservation in the laminar organization of cells and differences in somatic interactions across species. Our data revealed human-specific cell-cell proximity patterns and a markedly increased enrichment for interactions between neurons and non-neuronal cells in the human cortex.

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment.

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges' role in Giardia biology. Live imaging revealed that the flange grows to around 1 µm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia's unconventional actin cytoskeleton has an important role in supporting parasite attachment.

Versatile, do-it-yourself, low-cost spinning disk confocal microscope.

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (∼400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.

Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy.

Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid-Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy.

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells.

Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy.

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues.

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.

Volumetric, Nanoscale Optical Imaging of Mouse and Human Kidney via Expansion Microscopy.

Although light microscopy is a powerful tool for the assessment of kidney physiology and pathology, it has traditionally been unable to resolve structures separated by less than the ~250 nm diffraction limit of visible light. Here, we report on the optimization, validation, and application of a recently developed super-resolution fluorescence microscopy method, called expansion microscopy (ExM), for volumetric interrogation of mouse and human kidney tissue with 70-75 nm lateral and ~250 nm axial spatial resolution. Using ExM with a standard confocal microscope, we resolve fine details of structures that have traditionally required visualization by electron microscopy, including podocyte foot processes, the glomerular basement membrane, and the cytoskeleton. This inexpensive and accessible approach to volumetric, nanoscale imaging enables visualization of fine structural details of kidney tissues that were previously difficult or impossible to measure by conventional methodologies.

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM.

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization.

Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ∼65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ∼30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 , which localizes to disc microribbons, and the functionally unknown DAP16263 , which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.

The tetrameric kinesin Kif25 suppresses pre-mitotic centrosome separation to establish proper spindle orientation.

Microtubules tether centrosomes together during interphase. How this is accomplished and what benefit it provides to the cell is not known. We have identified a bipolar, minus-end-directed kinesin, Kif25, that suppresses centrosome separation. Kif25 is required to prevent premature centrosome separation during interphase. We show that premature centrosome separation leads to microtubule-dependent nuclear translocation, culminating in eccentric nuclear positioning that disrupts the cortical spindle positioning machinery. The activity of Kif25 during interphase is required to maintain a centred nucleus to ensure the spindle is stably oriented at the onset of mitosis.

Expansion microscopy with conventional antibodies and fluorescent proteins.

Expansion microscopy is a technique in which fluorophores on fixed specimens are linked to a swellable polymer that is physically expanded to enable super-resolution microscopy with ordinary microscopes. We have developed and characterized new methods for linking fluorophores to the polymer that now enable expansion microscopy with conventional fluorescently labeled antibodies and fluorescent proteins. Our methods simplify the procedure and expand the palette of compatible labels, allowing rapid dissemination of the technique.

Point by Point: An Introductory Guide to Sample Preparation for Single-Molecule, Super-Resolution Fluorescence Microscopy.

Single-molecule, localization-based, super resolution microscopy is able to reveal detailed subcellular structures and protein distributions below the classical ∼250-nm diffraction limit of light, but utilizing this technique effectively requires a combination of careful sample preparation, data acquisition, and data analysis, which can be daunting to novice researchers. In this protocol, detailed instructions on preparation of robust reference samples for super-resolution microscopy, including the cytoskeleton (microtubules), membrane-bound organelles (peroxisomes), and scaffold proteins (clathrin), are provided. These samples also constitute a representative subset of imaging targets of interest to biological researchers and highlight the differences and similarities in sample preparation.

Single-nanoparticle near-infrared surface plasmon resonance microscopy for real-time measurements of DNA hybridization adsorption.

A novel 814 nm near-infrared surface plasmon resonance (SPR) microscope is used for the real-time detection of the sequence-selective hybridization adsorption of single DNA-functionalized gold nanoparticles. The objective-coupled, high numerical aperture SPR microscope is capable of imaging in situ the adsorption of single polystyrene and gold particles with diameters ranging from 450 to 20 nm onto a 90 µm × 70 µm area of a gold thin film with a time resolution of approximately 1-3 s. Initial real-time SPR imaging (SPRI) measurements were performed to detect the accumulation of 40 nm gold nanoparticles for 10 min onto a gold thin film functionalized with a 100% complementary DNA surface at concentrations from 5 pM to 100 fM by counting individual particle binding events. A 100% noncomplementary DNA surface exhibited virtually no nanoparticle adsorption. In contrast, in a second set of SPRI measurements, two component complementary/noncomplementary mixed DNA monolayers that contained a very small percentage of complementary sequences ranging from 0.1 to 0.001%, showed both permanent and transient hybridization adsorption of the gold nanoparticles that could be tracked both temporally and spatially with the SPR microscope. These experiments demonstrate that SPR imaging measurements of single biofunctionalized nanoparticles can be incorporated into bioaffinity biosensing methods at subpicomolar concentrations.

Electrodeposition of polydopamine thin films for DNA patterning and microarrays.

The controlled electrodeposition of functional polydopamine (PDA) thin films from aqueous dopamine solutions is demonstrated with a combination of electrochemistry, atomic force microscopy (AFM), and surface plasmon resonance (SPR) measurements. PDA micropatterns are then fabricated by electrodeposition on micrometer length scale gold electrodes and used for attaching amino-modified single-stranded DNA (ssDNA). After hybridization with fluorescently labeled ssDNA, the fluorescence microscopy characterization reveals that: (i) PDA can be toposelectively deposited at the microscale and (ii) electrochemically deposited PDA can be functionalized with amino-terminated ssDNA using the same chemistry as that for spontaneously deposited PDA. Finally, the application of electrodeposited PDA thin films to fabricate ssDNA microarrays is reported using SPR imaging (SPRI) measurements for the detection of DNA and DNA-modified gold nanoparticles.

Fabrication of DNA microarrays on polydopamine-modified gold thin films for SPR imaging measurements.

Polydopamine (PDA) films were fabricated on thin film gold substrates in a single-step polymerization-deposition process from dopamine solutions and then employed in the construction of robust DNA microarrays for the ultrasensitive detection of biomolecules with nanoparticle-enhanced surface plasmon resonance (SPR) imaging. PDA multilayers with thicknesses varying from 1 to 5 nm were characterized with a combination of scanning angle SPR and AFM experiments, and 1.3 ± 0.2 nm PDA multilayers were chosen as an optimal thickness for the SPR imaging measurements. DNA microarrays were then fabricated by the reaction of amine-functionalized single-stranded DNA (ssDNA) oligonucleotides with PDA-modified gold thin film microarray elements, and were subsequently employed in SPR imaging measurements of DNA hybridization adsorption and protein-DNA binding. Concurrent control experiments with non-complementary ssDNA sequences demonstrated that the adhesive PDA multilayer was also able to provide good resistance to the nonspecific binding of biomolecules. Finally, a series of SPR imaging measurements of the hybridization adsorption of DNA-modified gold nanoparticles onto mixed sequence DNA microarrays were used to confirm that the use of PDA multilayer films is a simple, rapid, and versatile method for fabricating DNA microarrays for ultrasensitive nanoparticle-enhanced SPR imaging biosensing.

Lithographically patterned electrodeposition of gold, silver, and nickel nanoring arrays with widely tunable near-infrared plasmonic resonances.

A novel low-cost nanoring array fabrication method that combines the process of lithographically patterned nanoscale electrodeposition (LPNE) with colloidal lithography is described. Nanoring array fabrication was accomplished in three steps: (i) a thin (70 nm) sacrificial nickel or silver film was first vapor-deposited onto a plasma-etched packed colloidal monolayer; (ii) the polymer colloids were removed from the surface, a thin film of positive photoresist was applied, and a backside exposure of the photoresist was used to create a nanohole electrode array; (iii) this array of nanoscale cylindrical electrodes was then used for the electrodeposition of gold, silver, or nickel nanorings. Removal of the photoresist and sacrificial metal film yielded a nanoring array in which all of the nanoring dimensions were set independently: the inter-ring spacing was fixed by the colloidal radius, the radius of the nanorings was controlled by the plasma etching process, and the width of the nanorings was controlled by the electrodeposition process. A combination of scanning electron microscopy (SEM) measurements and Fourier transform near-infrared (FT-NIR) absorption spectroscopy were used to characterize the nanoring arrays. Nanoring arrays with radii from 200 to 400 nm exhibited a single strong NIR plasmonic resonance with an absorption maximum wavelength that varied linearly from 1.25 to 3.33 µm as predicted by a simple standing wave model linear antenna theory. This simple yet versatile nanoring array fabrication method was also used to electrodeposit concentric double gold nanoring arrays that exhibited multiple NIR plasmonic resonances.

On-chip synthesis of protein microarrays from DNA microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications.

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of mRNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and antiluciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.