Editorial Note to: In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections, but do not have single cell genomic resolution. Here, we developed a computational approach called SChart, that combines these two datasets to achieve single cell spatial mapping of cell types, cell states and continuous phenotypes. We applied SChart to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues to validate our approach. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied SChart to identify subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data shows that SChart can accurately map single cells in diverse tissue types to resolve their spatial organization into cellular neighborhoods and tissue structures.

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Highly Multiplexed Spatial Mapping of Microbial Communities

10.1101/678672 ◽

2019 ◽

Author(s):

Hao Shi ◽

Warren Zipfel ◽

Ilana Brito ◽

Iwijn De Vlaminck

Keyword(s):

Microbial Communities ◽

Spatial Organization ◽

Single Cells ◽

Cost Effective ◽

Probe Design ◽

E Coli ◽

Rich Diversity ◽

Effective Technology ◽

Phylogenetic Resolution

ABSTRACTMapping the complex biogeography of microbial communities in situ with high taxonomic and spatial resolution poses a major challenge because of the high density and rich diversity of species in environmental microbiomes and the limitations of optical imaging technology. Here, we introduce High Phylogenetic Resolution microbiome mapping by Fluorescence In-Situ Hybridization (HiPR-FISH), a versatile and cost-effective technology that uses binary encoding and spectral imaging and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. We demonstrate the ability of 10-bit HiPR-FISH to distinguish 1023 E. coli strains, each fluorescently labeled with a unique binary barcode. HiPR-FISH, in conjunction with custom algorithms for automated probe design and segmentation of single-cells in the native context of tissues, reveals the intricate spatial architectures formed by bacteria in the human oral plaque microbiome and disruption of spatial networks in the mouse gut microbiome in response to antibiotic treatment. HiPR-FISH provides a framework for analyzing the spatial organization of microbial communities in tissues and the environment at single cell resolution.

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Visualization of loop extrusion by DNA nanoscale tracing in single human cells

10.1101/2021.04.12.439407 ◽

2021 ◽

Author(s):

KS Beckwith ◽

Ø Ødegård-Fougner ◽

NR Morero ◽

C Barton ◽

F Schueder ◽

...

Keyword(s):

Chromatin Structure ◽

Spatial Organization ◽

Driving Forces ◽

Single Cells ◽

Human Cells ◽

Random Coil ◽

Loop Formation ◽

Chromosomal Dna ◽

3D Genome

SummaryThe spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensates and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random structures such as chromatin compartments and topologically associating domains2,3. However, if the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we present LoopTrace, a FISH workflow for high-resolution reconstruction of 3D genome architecture without DNA denaturation. Classical fluorescence in situ hybridization approaches can link chromatin architecture to DNA sequence but disrupt chromatin structure at the critical nanoscale of individual loops. Our method conserves chromatin structure and can resolve the 3D-fold of chromosomal DNA with better than 5-kb-resolution in single human cells. Our results show that the chromatin fiber behaves as a random coil that can be further structured in a manner consistent with loop formation, explaining the emergence of topologically associated domain-like features in cell population averages. Mining a large amount of single-cell data computationally, we reveal chromatin folding intermediates consistent with progressive loop extrusion and stabilized loops, highlighting the power of our method to visualize the nanoscale features of genome organization in situ.

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Probing the spatial organization of nucleic acids within cells by nonisotopic in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012045x ◽

1992 ◽

Vol 50 (1) ◽

pp. 10-11

Author(s):

Jeanne Bentley Lawrence

Keyword(s):

In Situ Hybridization ◽

Nucleic Acids ◽

Spatial Organization ◽

Experimental Approach ◽

Single Cells ◽

Potential Applications ◽

A Cell ◽

Differential Gene ◽

High Degree

In situ hybridization is a powerful experimental approach that directly couples molecular and cytological information in a visual context. Advances in hybridization procedures over recent years, coupled with previously described non-isotopic labelling methods developed in a number of laboratories, now provide a way to detect nucleic acids within cells with a high degree of resolution and sensitivity. Adaptations of this technology allow either DNA or RNA to be detected and visualized either with the light microscope, using fluorescence or colorimetric methods, or with the electron microscope using antibodies conjugated to gold or peroxidase. The potential applications of this technology are relevant to numerous areas of biomedical research and range from the more straightforward study of differential gene expression in single cells within a population to the precise localization of individual genes or RNAs within the cytoplasm or nucleus of a cell.

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Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

mBio ◽

10.1128/mbio.00796-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 49

Author(s):

William H. DePas ◽

Ruth Starwalt-Lee ◽

Lindsey Van Sambeek ◽

Sripriya Ravindra Kumar ◽

Viviana Gradinaru ◽

...

Keyword(s):

Cystic Fibrosis ◽

Growth Rate ◽

Microbial Communities ◽

Spatial Organization ◽

Single Cells ◽

Host Cells ◽

Individual Species ◽

Complex Environments ◽

Host Interactions

ABSTRACT Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus , Pseudomonas aeruginosa , Streptococcus sp., and Achromobacter xylosoxidans , we demonstrate a spectrum of aggregation states: from mostly single cells ( A. xylosoxidans ), to medium-sized clusters ( S. aureus ), to a mixture of single cells and large aggregates ( P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum. IMPORTANCE The advent of metagenomic and metatranscriptomic analyses has improved our understanding of microbial communities by empowering us to identify bacteria, calculate their abundance, and profile gene expression patterns in complex environments. We are still technologically limited, however, in regards to the many questions that bulk measurements cannot answer, specifically in assessing the spatial organization of microbe-microbe and microbe-host interactions. Here, we demonstrate the power of an enhanced optical clearing method, MiPACT, to survey important aspects of bacterial physiology (aggregation, host interactions, and growth rate), in situ , with preserved spatial information when coupled to rRNA detection by HCR. Our application of MiPACT-HCR to cystic fibrosis patient sputum revealed species-specific aggregation patterns, yet slow growth characterized the vast majority of bacterial cells regardless of their cell type. More broadly, MiPACT, coupled with fluorescent labeling, promises to advance the direct study of microbial communities in diverse environments, including microbial habitats within mammalian systems.

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Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

10.26434/chemrxiv.12046125 ◽

2020 ◽

Author(s):

Feifei Jia ◽

Jie Wang ◽

Yanyan Zhang ◽

Qun Luo ◽

Luyu Qi ◽

...

Keyword(s):

Mass Spectrometry ◽

Secondary Ion Mass Spectrometry ◽

Single Cells ◽

Time Of Flight ◽

E Coli ◽

Tof Sims ◽

Ion Mass Spectrometry ◽

Chemical Tags ◽

Secondary Ion

In situ visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for in situ visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.

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In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

ISTFA 1999: Conference Proceedings from the 25th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa1999p0311 ◽

1999 ◽

Author(s):

Gunnar Zimmermann ◽

Richard Chapman

Keyword(s):

Electron Beam ◽

Single Cell ◽

Single Cells ◽

Ion Beam ◽

Gate Oxide ◽

Ion Milling ◽

Dual Beam ◽

Sem Imaging ◽

Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara < 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

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Faculty Opinions recommendation of Synthetic recording and in situ readout of lineage information in single cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727004367.793527019 ◽

2017 ◽

Author(s):

Steve Brown

Keyword(s):

Single Cells

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Label-Free and Quantitative Dry Mass Monitoring for Single Cells during In Situ Culture

Cells ◽

10.3390/cells10071635 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1635

Author(s):

Ya Su ◽

Rongxin Fu ◽

Wenli Du ◽

Han Yang ◽

Li Ma ◽

...

Keyword(s):

Cell Growth ◽

Single Cell ◽

Hela Cells ◽

Quantitative Measurement ◽

Single Cells ◽

Dry Mass ◽

Quantitative Detection ◽

Label Free ◽

Mass Sensitivity

Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.

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