Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions.

Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.

Molecular robotic agents that survey molecular landscapes for information retrieval.

DNA-based artificial motors have allowed the recapitulation of biological functions and the creation of new features. Here, we present a molecular robotic system that surveys molecular environments and reports spatial information in an autonomous and repeated manner. A group of molecular agents, termed 'crawlers', roam around and copy information from DNA-labeled targets, generating records that reflect their trajectories. Based on a mechanism that allows random crawling, we show that our system is capable of counting the number of subunits in example molecular complexes. Our system can also detect multivalent proximities by generating concatenated records from multiple local interactions. We demonstrate this capability by distinguishing colocalization patterns of three proteins inside fixed cells under different conditions. These mechanisms for examining molecular landscapes may serve as a basis towards creating large-scale detailed molecular interaction maps inside the cell with nanoscale resolution.

SpatialData: an open and universal data framework for spatial omics.

Spatially resolved omics technologies are transforming our understanding of biological tissues. However, the handling of uni- and multimodal spatial omics datasets remains a challenge owing to large data volumes, heterogeneity of data types and the lack of flexible, spatially aware data structures. Here we introduce SpatialData, a framework that establishes a unified and extensible multiplatform file-format, lazy representation of larger-than-memory data, transformations and alignment to common coordinate systems. SpatialData facilitates spatial annotations and cross-modal aggregation and analysis, the utility of which is illustrated in the context of multiple vignettes, including integrative analysis on a multimodal Xenium and Visium breast cancer study.

A perspective on FAIR quality control in multiplexed imaging data processing.

Multiplexed imaging approaches are getting increasingly adopted for imaging of large tissue areas, yielding big imaging datasets both in terms of the number of samples and the size of image data per sample. The processing and analysis of these datasets is complex owing to frequent technical artifacts and heterogeneous profiles from a high number of stained targets To streamline the analysis of multiplexed images, automated pipelines making use of state-of-the-art algorithms have been developed. In these pipelines, the output quality of one processing step is typically dependent on the output of the previous step and errors from each step, even when they appear minor, can propagate and confound the results. Thus, rigorous quality control (QC) at each of these different steps of the image processing pipeline is of paramount importance both for the proper analysis and interpretation of the analysis results and for ensuring the reusability of the data. Ideally, QC should become an integral and easily retrievable part of the imaging datasets and the analysis process. Yet, limitations of the currently available frameworks make integration of interactive QC difficult for large multiplexed imaging data. Given the increasing size and complexity of multiplexed imaging datasets, we present the different challenges for integrating QC in image analysis pipelines as well as suggest possible solutions that build on top of recent advances in bioimage analysis.

Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Multiplexed fluorescence imaging is typically limited to three- to five-plex on standard setups. Sequential imaging methods based on iterative labeling and imaging enable practical higher multiplexing, but generally require a complex fluidic setup with several rounds of slow buffer exchange (tens of minutes to an hour for each exchange step). We report the thermal-plex method, which removes complex and slow buffer exchange steps and provides fluidic-free, rapid sequential imaging. Thermal-plex uses simple DNA probes that are engineered to fluoresce sequentially when, and only when, activated with transient exposure to heating spikes at designated temperatures (thermal channels). Channel switching is fast (<30 s) and is achieved with a commercially available and affordable on-scope heating device. We demonstrate 15-plex RNA imaging (five thermal × three fluorescence channels) in fixed cells and retina tissues in less than 4 min, without using buffer exchange or fluidics. Thermal-plex introduces a new labeling method for efficient sequential multiplexed imaging.

Enablers and challenges of spatial omics, a melting pot of technologies.

Spatial omics has emerged as a rapidly growing and fruitful field with hundreds of publications presenting novel methods for obtaining spatially resolved information for any omics data type on spatial scales ranging from subcellular to organismal. From a technology development perspective, spatial omics is a highly interdisciplinary field that integrates imaging and omics, spatial and molecular analyses, sequencing and mass spectrometry, and image analysis and bioinformatics. The emergence of this field has not only opened a window into spatial biology, but also created multiple novel opportunities, questions, and challenges for method developers. Here, we provide the perspective of technology developers on what makes the spatial omics field unique. After providing a brief overview of the state of the art, we discuss technological enablers and challenges and present our vision about the future applications and impact of this melting pot.

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

Concurrent inhibition of CDK2 adds to the anti-tumour activity of CDK4/6 inhibition in GIST.

BACKGROUND: Advanced gastrointestinal stromal tumour (GIST) is characterised by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximising response to therapeutic CDK4/6 inhibition. METHODS: Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples. The impact of inhibitors of CDK2, CDK4 and CDK2/4/6 was determined through cell proliferation and protein detection assays. CDK-inhibitor resistance mechanisms were characterised in GIST cell lines after long-term exposure. RESULTS: We identify recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways in clinical GIST samples. Therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation. Moreover, RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) are mechanisms of acquired CDK-inhibitor resistance in GIST. CONCLUSIONS: These studies establish the biological rationale for CDK2 and CDK4/6 co-inhibition as a therapeutic strategy in patients with advanced GIST, including metastatic GIST progressing on tyrosine kinase inhibitors.

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA.

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 µL reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

Super-resolution labelling with Action-PAINT.

Recent advances in localization-based super-resolution microscopy have enabled researchers to visualize single molecular features down to individual molecular components (~5 nm), but do not yet allow manipulation of single-molecule targets in a user-prescribed, context-dependent manner. Here we report an 'Action-PAINT' (PAINT, point accumulation for imaging in nanoscale topography) strategy for super-resolution labelling upon visualization on single molecules. This approach monitors and localizes DNA binding events in real time with DNA-PAINT, and upon visualization of binding to a desired location, photo-crosslinks the DNA to affix the molecular label. We showed the efficiency of 3-cyanovinylcarbazole nucleoside photo-inducible crosslinking on single molecular targets and developed a software package for real-time super-resolution imaging and crosslinking control. We then benchmarked our super-resolution labelling method on synthetic DNA nanostructures and demonstrated targeted multipoint labelling on various complex patterns with 30 nm selectivity. Finally, we performed targeted in situ labelling on fixed microtubule samples with a 40 nm target size and custom-controlled, subdiffraction spacing.

Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues.

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reaction (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrate 5- to 180-fold signal amplification in diverse samples (cultured cells, cryosections, formalin-fixed paraffin-embedded sections and whole-mount tissues), as well as simultaneous signal amplification for ten different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.

SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues.

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.

OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes.

Oligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.

Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT.

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.

Mapping molecules in scanning far-field fluorescence nanoscopy.

In fluorescence microscopy, the distribution of the emitting molecule number in space is usually obtained by dividing the measured fluorescence by that of a single emitter. However, the brightness of individual emitters may vary strongly in the sample or be inaccessible. Moreover, with increasing (super-) resolution, fewer molecules are found per pixel, making this approach unreliable. Here we map the distribution of molecules by exploiting the fact that a single molecule emits only a single photon at a time. Thus, by analysing the simultaneous arrival of multiple photons during confocal imaging, we can establish the number and local brightness of typically up to 20 molecules per confocal (diffraction sized) recording volume. Subsequent recording by stimulated emission depletion microscopy provides the distribution of the number of molecules with subdiffraction resolution. The method is applied to mapping the three-dimensional nanoscale organization of internalized transferrin receptors on human HEK293 cells.

A contamination-insensitive probe for imaging specific biomolecules by secondary ion mass spectrometry.

Imaging techniques should differentiate between specific signals, from the biomolecules of interest, and non-specific signals, from the background. We present a probe containing (15)N and (14)N isotopes in approximately equal proportion, for secondary ion mass spectrometry imaging. This probe designed for a precise biomolecule analysis is insensitive to background signals.

Secondary-ion mass spectrometry of genetically encoded targets.

Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added (19) F-enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The (19) F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell-culture systems, as well as small model organisms.

Multi-protein assemblies underlie the mesoscale organization of the plasma membrane.

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity.

Correlated optical and isotopic nanoscopy.

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes ((15)N, (13)C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.