Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on ...DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.
Multiparameter tissue imaging enables analysis of cell‐cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to ...biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO‐Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a LN. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single‐cell analysis on the data, including unsupervised clustering, that revealed 31 cell types across all tissues. We compared cell‐type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell‐cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre‐existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging.
We simplified the multiplexed imaging method CO‐Detection by indEXing (CODEX) by employing cyclic stripping and annealing of fluorescent oligonucleotides to DNA‐barcoded antibodies. We stained five human lymphoid tissues with a panel of 46 antibodies. This enabled quantitative spatial single‐cell biology analysis of differential cell‐type composition of immune follicles.
The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall ...health
. The intesting has a length of over nine metres, along which there are differences in structure and function
. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.
The model and tool used for information presentation is critical to effective pedagogy. Slide-based model limits information to a linear sequence. In the alternative, canvas-based paradigm, ...information is laid out in space and depth, and the presentation flows as a view port makes transitions such as pan, zoom, and call-out on this three dimensional representation of information. We have implemented CANSCAPE, a canvas-based presentation editor with a web interface, using open standards such as Scalable Vector Graphics (SVG) and JavaScript, and using a web browser as the presentation viewer.
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