ArticleLaboratory investigation; a journal of technical methods and pathology2024
Increased Multiplexity in Optical Tissue Clearing-Based Three-Dimensional Immunofluorescence Microscopy of the Tumor Microenvironment by Light-Emitting Diode Photobleaching.
Article in Laboratory investigation; a journal of technical methods and pathology, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- NicheProt: Cell-type-resolved proteomics of tissue compartments.Science advances · 2026Article
- Review
- Human liver and pancreas innervation: resolving 3D neurohistological challenges and advancing insights.Journal of biomedical science · 2025Review
- Article
- An open-source photobleacher for fluorescence imaging of large pigment-rich tissues.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Unlocking the potential of large-scale 3D imaging with tissue clearing techniques.Microscopy (Oxford, England) · 2025Review
- Article
- Spatial proteomics of the tumor microenvironment in melanoma: current insights and future directions.Frontiers in immunology · 2025Review
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6 authors.
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Abstract
Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 μm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.
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