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Precise immuno-fluorescence canceling enables highly multiplexed imaging [RNA-seq]

ABSTRACT: Precise immuno-fluorescence canceling enables highly multiplexed imaging [RNA-seq]

PROVIDER: PRJNA1015232 | ENA |

REPOSITORIES: ENA

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Precise immuno-fluorescence canceling enables highly multiplexed imaging

Project description:Precise immuno-fluorescence canceling enables highly multiplexed imaging

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Precise immuno-fluorescence canceling enables highly multiplexed imaging [RNA-seq]

Project description:Cell states are regulated by extrinsic signals from various external factors such as intercellular interactions, and intrinsic gene expression. Although comprehensive cell state profiling has been attempted, it remains simultaneous analysis of signal activation has still been challenging. Multiplexed imaging is a technique acquiring multiple protein information at a single cell level as traditional immunofluorescence. However, the method often compromises resolution, hindering the analysis of intracellular localization dynamics and post-translational modifications of proteins. To address these limitations, we developed an erasable fluorescence method using disulfide linkers to label antibodies. We term these antibodies ‘Precise Emission Canceling Antibodies (PECAbs)’. PECAb allows for high-resolution iterative imaging with minimal non-specific binding. Automation enables our system to achieve reproducible quantitative analysis using 206 antibodies. The resulting quantitative data allow reconstruction of the spatiotemporal dynamics of signaling pathways over both long and short timescales. Additionally, combining this approach with sequential RNA-FISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system serves as a comprehensive platform for analyzing complex cell processes, from signal transduction to gene expression.

2024-04-21 | GSE242886 | GEO

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Precise immuno-fluorescence canceling enables highly multiplexed imaging

Project description:This SuperSeries is composed of the SubSeries listed below.

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Wound assessment using a point of care bacterial fluorescence imaging device in dogs and cats

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Multiplexed protein and drug imaging by super-resolution ion beam imaging

Project description:Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the “tag” and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.

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Project description:Multiplexed imaging of nucleome architectures in single cells of mammalian tissue

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