3D Optical Tissue Microscopy Towards Spatial Cell Proteomics

Spatial proteomics is an emerging field that enables the study of protein expression and organization within complex tissue environments. This dissertation presented two novel spatial proteomics methodologies to advance tissue-scale and cell-type-specific protein profiling through 3D optical microscopy. The first approach integrated light-sheet fluorescence microscopy (LSFM) and confocal laser scanning microscopy (CLSM) into a multi-scale imaging pipeline. A novel tissue-clearing and rehydration method was developed to enhance tissue transparency and preserve antigenicity, allowing for sequential imaging at organ-, tissue-, and cellular-level resolutions. This workflow was applied to an asthma mouse model, revealing distinct spatial distributions of immune cell infiltrates in the lung and their relationship to anatomical structures such as airways and blood vessels. The second approach introduced a tissue-niche-based and cell-type-selective proteomics strategy (Niche&CellType-SP). This method employed microscopic photobleaching to encode spatial information into single cells within thick tissue slices. Labeled cells are subsequently dissociated, sorted via flow cytometry, and analyzed through liquid chromatography-mass spectrometry (LC-MS) for deep proteomic profiling. This technique was validated in mouse models of systemic inflammation and peripheral T cell lymphoma (PTCL), identifying distinct dendritic cell subpopulations and exploring T cell-macrophage interactions. The workflow was optimized for human tumor specimens to translate these methods into clinical applications. Future improvements will focus on incorporating antigen retrieval for formalin-fixed tissues, enhancing tissue dissociation efficiency, immune cell enrichment, and preserving fluorescence and antibody bonding to facilitate the translation to biomarker discovery and personalized medicine in clinics. This dissertation advances the field of spatial proteomics by introducing two innovative optical microscopy tools that enable multi-resolution observation, cell-type-specific protein mapping, and comprehensive protein profiling. These methodologies offer valuable insights into identifying novel immune cell subtypes, screening protein biomarkers as potential therapeutic targets, investigating macrophage influence on tumor T-cell progression, and exploring ECM-driven macrophage differentiation in tumors. Together, these approaches lay the foundation for future biomedical research, immunology, and translational medicine applications.