Author Correction: Highly specific multiplexed RNA imaging in tissues with split-FISH.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly specific multiplexed RNA imaging in tissues with split-FISH.

We present split-FISH, a multiplexed fluorescence in situ hybridization method that leverages a split-probe design to achieve enhanced specificity. Split-FISH reduces off-target background fluorescence, decreases false positives and enables accurate RNA profiling in uncleared tissues. We demonstrate the efficacy of split-FISH on various mouse tissues by quantifying the distribution and abundance of 317 genes in single cells and reveal diverse localization patterns for spatial regulation of the transcriptome in complex tissues.

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization.

Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.

Highly sensitive spatial transcriptomics using FISHnCHIPs of multiple co-expressed genes.

High-dimensional, spatially resolved analysis of intact tissue samples promises to transform biomedical research and diagnostics, but existing spatial omics technologies are costly and labor-intensive. We present Fluorescence In Situ Hybridization of Cellular HeterogeneIty and gene expression Programs (FISHnCHIPs) for highly sensitive in situ profiling of cell types and gene expression programs. FISHnCHIPs achieves this by simultaneously imaging ~2-35 co-expressed genes (clustered into modules) that are spatially co-localized in tissues, resulting in similar spatial information as single-gene Fluorescence In Situ Hybridization (FISH), but with ~2-20-fold higher sensitivity. Using FISHnCHIPs, we image up to 53 modules from the mouse kidney and mouse brain, and demonstrate high-speed, large field-of-view profiling of a whole tissue section. FISHnCHIPs also reveals spatially restricted localizations of cancer-associated fibroblasts in a human colorectal cancer biopsy. Overall, FISHnCHIPs enables fast, robust, and scalable cell typing of tissues with normal physiology or undergoing pathogenesis.

BANKSY unifies cell typing and tissue domain segmentation for scalable spatial omics data analysis.

Spatial omics data are clustered to define both cell types and tissue domains. We present Building Aggregates with a Neighborhood Kernel and Spatial Yardstick (BANKSY), an algorithm that unifies these two spatial clustering problems by embedding cells in a product space of their own and the local neighborhood transcriptome, representing cell state and microenvironment, respectively. BANKSY's spatial feature augmentation strategy improved performance on both tasks when tested on diverse RNA (imaging, sequencing) and protein (imaging) datasets. BANKSY revealed unexpected niche-dependent cell states in the mouse brain and outperformed competing methods on domain segmentation and cell typing benchmarks. BANKSY can also be used for quality control of spatial transcriptomics data and for spatially aware batch effect correction. Importantly, it is substantially faster and more scalable than existing methods, enabling the processing of millions of cell datasets. In summary, BANKSY provides an accurate, biologically motivated, scalable and versatile framework for analyzing spatially resolved omics data.

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection.

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.

Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single-molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single-molecule fluorescence measurements of biomacromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions in equilibrium with the initial buffer condition. To permeabilize the barrier, Staphylococcus aureus toxin α-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single-molecule fluorescence resonance energy transfer signals from vesicle-encapsulated guanine-rich DNA that folds in a G-quadruplex motif as well as from the Rep helicase-DNA system. We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17-37 °C). Ions can efficiently pass through preformed aHL channels to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report a 35-fold improvement in the aHL channel formation efficiency, making this approach more practical for wider applications. Finally, the temperature-dependent single-molecule enzymatic study inside these nanocontainers is demonstrated by measuring the Rep helicase repetitive shuttling dynamics along a single-stranded DNA at various temperatures. The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high-throughput measurements and sensors beyond the single-molecule fluorescence measurements.

Author Correction: Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors.

In the version of the article published, the author list is not accurate. Igor Cima and Min-Han Tan should have been authors, appearing after Mark Wong in the author list, while Paul Jongjoon Choi should not have been listed as an author. Igor Cima and Min-Han Tan both have the affiliation Institute of Bioengineering and Nanotechnology, Singapore, Singapore, and their contributions should have been noted in the Author Contributions section as "I.C. preprocessed Primary Cell Atlas data with inputs from M.-H.T." The following description of the contribution of Paul Jongjoon Choi should not have appeared: "P.J.C. supported the smFISH experiments." In the 'RCA: global panel' section of the Online Methods, the following sentence should have appeared as the second sentence, "An expression atlas of human primary cells (the Primary Cell Atlas) was preprocessed similarly to in ref. 55," with new reference 55 (Cima, I. et al. Tumor-derived circulating endothelial cell clusters in colorectal cancer. Science Transl. Med. 8, 345ra89, 2016).

Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors.

Intratumoral heterogeneity is a major obstacle to cancer treatment and a significant confounding factor in bulk-tumor profiling. We performed an unbiased analysis of transcriptional heterogeneity in colorectal tumors and their microenvironments using single-cell RNA-seq from 11 primary colorectal tumors and matched normal mucosa. To robustly cluster single-cell transcriptomes, we developed reference component analysis (RCA), an algorithm that substantially improves clustering accuracy. Using RCA, we identified two distinct subtypes of cancer-associated fibroblasts (CAFs). Additionally, epithelial-mesenchymal transition (EMT)-related genes were found to be upregulated only in the CAF subpopulation of tumor samples. Notably, colorectal tumors previously assigned to a single subtype on the basis of bulk transcriptomics could be divided into subgroups with divergent survival probability by using single-cell signatures, thus underscoring the prognostic value of our approach. Overall, our results demonstrate that unbiased single-cell RNA-seq profiling of tumor and matched normal samples provides a unique opportunity to characterize aberrant cell states within a tumor.

RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.

Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~10(4) to 10(6) pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.

Understanding oligonucleotide-templated nanocrystals: growth mechanisms and surface properties.

We describe studies of nanoparticle synthesis using oligonucleotides as capping ligands. The oligonucleotides nucleate, grow, and stabilize near-infrared fluorescent, approximately uniform PbS nanocrystals in an aqueous environment. The properties of the resulting particles strongly depend upon the sequences as well as synthesis conditions. Fourier Transform infrared measurements suggest that functional groups on the nucleobases such as carbonyl and amine moieties are responsible for surface passivation, while the phosphate backbone is strained to accommodate nucleobase bonding, preventing irreversible aggregation and thereby stabilizing the colloids. Our theoretical model indicates that oligonucleotide-mediated particle growth relies on the chemical reactivity of the oligonucleotide ligands that saturate dangling bonds of growing clusters, and favorable sequences are those that have the highest surface reactivity with growing particles. The oligonucleotide template approach is facile and versatile, offering a route to produce a range of material compositions for other chalcogenide semiconductor quantum dots and metal oxide nanoparticles.

Wide-field single metal nanoparticle spectroscopy for high throughput localized surface plasmon resonance sensing.

Noble metal nanoparticles (mNPs) have a distinct extinction spectrum arising from their ability to support Localized Surface Plasmon Resonance (LSPR). Single-particle biosensing with LSPR is label free and offers a number of advantages, including single molecular sensitivity, multiplex detection, and in vivo quantification of chemical species etc. In this article, we introduce Single-particle LSPR Imaging (SLI), a wide-field spectral imaging method for high throughput LSPR biosensing. The SLI utilizes a transmission grating to generate the diffraction spectra from multiple mNPs, which are captured using a Charge Coupled Device (CCD). With the SLI, we are able to simultaneously image and track the spectral changes of up to 50 mNPs in a single (∼1 s) exposure and yet still retain a reasonable spectral resolution for biosensing. Using the SLI, we could observe spectral shift under different local refractive index environments and demonstrate biosensing using biotin-streptavidin as a model system. To the best of our knowledge, this is the first time a transmission grating based spectral imaging approach has been used for mNPs LSPR sensing. The higher throughput LSPR sensing, offered by SLI, opens up a new possibility of performing label-free, single-molecule experiments in a high-throughput manner.

Dispersion of single-walled carbon nanotubes with an extended diazapentacene derivative.

The dispersion of SWCNTs by using a novel diazapentacene derivative is reported. The proposed pi-pi interactions between the diazapentacene derivative and SWCNTs leave their inherent properties virtually intact, as observed by several photophysical measurements. This approach is very attractive for manipulation of SWCNTs for electronic applications.

Aptamer-capped nanocrystal quantum dots: a new method for label-free protein detection.

We demonstrate that aptamer-capped near-infrared PbS quantum dots (QDs) can detect a target protein based on selective charge transfer. The water-soluble QDs are synthesized with the thrombin-binding aptamer, which retains the secondary quadruplex structure necessary for binding to thrombin. These QDs have diameters of 3-6 nm and fluoresce around 1050 nm. When the aptamer-functionalized QD binds to its target, a fluorescence quenching occurs due to charge transfer from amine groups on the protein to the QD. Thrombin is detected within 1 min with a detection limit of approximately 1 nM. This selective detection is observed even in the presence of high background concentrations of interfering negatively or positively charged proteins, suggesting that aptamer-capped QDs could be useful for label-free protein assays.