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Deep-tissue transcriptomics and subcellular imaging at high spatial resolution.

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Title: Deep-tissue transcriptomics and subcellular imaging at high spatial resolution.
Authors: Gandin, Valentina, Kim, Jun, Yang, Liang-Zhong, Lian, Yumin, Kawase, Takashi, Hu, Amy, Rokicki, Konrad, Fleishman, Greg, Tillberg, Paul, Castrejon, Alejandro Aguilera, Stringer, Carsen, Preibisch, Stephan, Liu, Zhe J.
Source: Science; 4/18/2025, Vol. 388 Issue 6744, p1-13, 13p
Subject Terms: BIOLOGICAL specimen analysis, CELL imaging, GENETIC barcoding, TRANSCRIPTOMES, EXPANSION microscopy, RNA analysis
Abstract: Limited color channels in fluorescence microscopy have long constrained spatial analysis in biological specimens. We introduce cycle hybridization chain reaction (cycleHCR), a method that integrates multicycle DNA barcoding with HCR to overcome this limitation. cycleHCR enables highly multiplexed imaging of RNA and proteins using a unified barcode system. Whole-embryo transcriptomics imaging achieved precise three-dimensional gene expression and cell fate mapping across a specimen depth of ~310 μm. When combined with expansion microscopy, cycleHCR revealed an intricate network of 10 subcellular structures in mouse embryonic fibroblasts. In mouse hippocampal slices, multiplex RNA and protein imaging uncovered complex gene expression gradients and cell-type–specific nuclear structural variations. cycleHCR provides a quantitative framework for elucidating spatial regulation in deep tissue contexts for research and has potential diagnostic applications. Editor's summary: The ability to image cells and detect the expression of specific RNAs and proteins in situ is critical for advancing our understanding of various biological processes. Recent advances in multiomic imaging have been constrained by the lack of availability of labeling dyes with distinct spectra and by other technical limitations. To address some of these challenges, Gandin et al. developed a method based on split hybridization chain reaction techniques using paired probes that must both recognize their targets to produce a positive signal. This approach allowed for the accurate detection of both rare and high-abundance targets. It can even work in thick tissue samples, which are difficult to study but helpful for examining cells in their biological context. The authors applied their method to uncover specific structures and gene expression patterns in mouse hippocampal slices and intact embryos. —Yevgeniya Nusinovich INTRODUCTION: Understanding how gene expression and subcellular structures are spatially organized within tissues is fundamental to biology and disease research. However, conventional fluorescence imaging is limited by color channels, restricting the simultaneous visualization of multiple molecular components. Although in situ spatial transcriptomics has expanded molecular imaging capabilities, it struggles to resolve high-abundance targets and dense cellular structures in thick specimens. Additionally, existing methods often require complex processing steps or suffer from signal loss in deeper regions. To overcome these challenges, we developed cycle hybridization chain reaction (HCR), a highly multiplexed RNA and protein imaging method that enables high-resolution transcriptomics and subcellular structure imaging in thick tissues. RATIONALE: Existing spatial transcriptomics approaches often rely on cross-round barcoding, which requires precise spot registration across imaging cycles. These techniques are generally limited to thin tissue sections and are prone to errors in dense molecular environments. Conversely, single-shot signal amplification methods such as HCR amplification excel at deep-tissue imaging of both sparse and dense targets; however, they lack high-throughput capacity. To address these limitations, we developed cycleHCR, which integrates multicycle DNA barcoding with HCR amplification. This approach enables high-throughput three-dimensional (3D) spatial analysis of RNA and protein distributions in thick tissue specimens, facilitating precise molecular mapping across multiple spatial scales. RESULTS: We applied cycleHCR to whole-mount mouse embryo transcriptomics, imaging 254 lineage-specific genes in E6.5-7.0 embryos. By integrating automated imaging and computational pipelines, we achieved precise 3D gene expression and cell fate mapping across a specimen depth of ~310 µm. Cluster analysis of single-cell gene expression data identified nine distinct cell populations, corresponding to known developmental lineages, with well-defined spatial organization. Our data enabled spatial analysis of gene expression gradients and heterogeneity, providing single-cell resolution insights into how gene expression varies across embryonic structures. Beyond transcriptomics, cycleHCR was combined with expansion microscopy to visualize 10 distinct subcellular structures in mouse embryonic fibroblasts. We observed complex nuclear and cytoplasmic architectures with enhanced spatial resolution. Moreover, the platform enabled unified multiplex RNA and protein imaging in mouse hippocampal slices, uncovering intricate gene expression gradients and cell-type–specific nuclear structural variations. CONCLUSION: By overcoming the limitations of traditional fluorescence imaging and existing spatial transcriptomics methods, cycleHCR offers key advantages: (i) Single-shot imaging enables robust deep-tissue in situ transcriptomics for both sparse and dense targets. (ii) The resulting empirical images allow immediate validation and visualization of molecular targets. (iii) A barcode system supports joint RNA and protein imaging, enabling cross-modality spatial analysis of cell types and subcellular organization. As a scalable spatial omics platform, cycleHCR is poised to advance developmental biology, neuroscience, and systems biology. Its adaptability across different specimen types highlights its potential as a next-generation imaging tool for deciphering the spatial arrangement of molecular components within intact tissues. Further optimization of microscopy modalities and sample preparation will enhance imaging depth and throughput, with promising applications in disease research and precision diagnostics. Unified multiplex RNA and protein imaging through cycleHCR.: Samples are first labeled with antibody and probe pools, where each target is uniquely barcoded with a split left (L) and right (R) barcode. The cycleHCR automation pipeline then reads targets using distinct L + R readout probes equipped with split HCR initiators. Signals are amplified through HCR, enabling deep tissue detection. After volumetric imaging, readout probes are instantly erased using chemical treatment, allowing for the next round of detection. Arrowheads indicate the 3′ end of DNA or RNA. [ABSTRACT FROM AUTHOR]
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Abstract:Limited color channels in fluorescence microscopy have long constrained spatial analysis in biological specimens. We introduce cycle hybridization chain reaction (cycleHCR), a method that integrates multicycle DNA barcoding with HCR to overcome this limitation. cycleHCR enables highly multiplexed imaging of RNA and proteins using a unified barcode system. Whole-embryo transcriptomics imaging achieved precise three-dimensional gene expression and cell fate mapping across a specimen depth of ~310 μm. When combined with expansion microscopy, cycleHCR revealed an intricate network of 10 subcellular structures in mouse embryonic fibroblasts. In mouse hippocampal slices, multiplex RNA and protein imaging uncovered complex gene expression gradients and cell-type–specific nuclear structural variations. cycleHCR provides a quantitative framework for elucidating spatial regulation in deep tissue contexts for research and has potential diagnostic applications. Editor's summary: The ability to image cells and detect the expression of specific RNAs and proteins in situ is critical for advancing our understanding of various biological processes. Recent advances in multiomic imaging have been constrained by the lack of availability of labeling dyes with distinct spectra and by other technical limitations. To address some of these challenges, Gandin et al. developed a method based on split hybridization chain reaction techniques using paired probes that must both recognize their targets to produce a positive signal. This approach allowed for the accurate detection of both rare and high-abundance targets. It can even work in thick tissue samples, which are difficult to study but helpful for examining cells in their biological context. The authors applied their method to uncover specific structures and gene expression patterns in mouse hippocampal slices and intact embryos. —Yevgeniya Nusinovich INTRODUCTION: Understanding how gene expression and subcellular structures are spatially organized within tissues is fundamental to biology and disease research. However, conventional fluorescence imaging is limited by color channels, restricting the simultaneous visualization of multiple molecular components. Although in situ spatial transcriptomics has expanded molecular imaging capabilities, it struggles to resolve high-abundance targets and dense cellular structures in thick specimens. Additionally, existing methods often require complex processing steps or suffer from signal loss in deeper regions. To overcome these challenges, we developed cycle hybridization chain reaction (HCR), a highly multiplexed RNA and protein imaging method that enables high-resolution transcriptomics and subcellular structure imaging in thick tissues. RATIONALE: Existing spatial transcriptomics approaches often rely on cross-round barcoding, which requires precise spot registration across imaging cycles. These techniques are generally limited to thin tissue sections and are prone to errors in dense molecular environments. Conversely, single-shot signal amplification methods such as HCR amplification excel at deep-tissue imaging of both sparse and dense targets; however, they lack high-throughput capacity. To address these limitations, we developed cycleHCR, which integrates multicycle DNA barcoding with HCR amplification. This approach enables high-throughput three-dimensional (3D) spatial analysis of RNA and protein distributions in thick tissue specimens, facilitating precise molecular mapping across multiple spatial scales. RESULTS: We applied cycleHCR to whole-mount mouse embryo transcriptomics, imaging 254 lineage-specific genes in E6.5-7.0 embryos. By integrating automated imaging and computational pipelines, we achieved precise 3D gene expression and cell fate mapping across a specimen depth of ~310 µm. Cluster analysis of single-cell gene expression data identified nine distinct cell populations, corresponding to known developmental lineages, with well-defined spatial organization. Our data enabled spatial analysis of gene expression gradients and heterogeneity, providing single-cell resolution insights into how gene expression varies across embryonic structures. Beyond transcriptomics, cycleHCR was combined with expansion microscopy to visualize 10 distinct subcellular structures in mouse embryonic fibroblasts. We observed complex nuclear and cytoplasmic architectures with enhanced spatial resolution. Moreover, the platform enabled unified multiplex RNA and protein imaging in mouse hippocampal slices, uncovering intricate gene expression gradients and cell-type–specific nuclear structural variations. CONCLUSION: By overcoming the limitations of traditional fluorescence imaging and existing spatial transcriptomics methods, cycleHCR offers key advantages: (i) Single-shot imaging enables robust deep-tissue in situ transcriptomics for both sparse and dense targets. (ii) The resulting empirical images allow immediate validation and visualization of molecular targets. (iii) A barcode system supports joint RNA and protein imaging, enabling cross-modality spatial analysis of cell types and subcellular organization. As a scalable spatial omics platform, cycleHCR is poised to advance developmental biology, neuroscience, and systems biology. Its adaptability across different specimen types highlights its potential as a next-generation imaging tool for deciphering the spatial arrangement of molecular components within intact tissues. Further optimization of microscopy modalities and sample preparation will enhance imaging depth and throughput, with promising applications in disease research and precision diagnostics. Unified multiplex RNA and protein imaging through cycleHCR.: Samples are first labeled with antibody and probe pools, where each target is uniquely barcoded with a split left (L) and right (R) barcode. The cycleHCR automation pipeline then reads targets using distinct L + R readout probes equipped with split HCR initiators. Signals are amplified through HCR, enabling deep tissue detection. After volumetric imaging, readout probes are instantly erased using chemical treatment, allowing for the next round of detection. Arrowheads indicate the 3′ end of DNA or RNA. [ABSTRACT FROM AUTHOR]
ISSN:00368075
DOI:10.1126/science.adq2084