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Background Imaging Mass Cytometry™ (IMC™) technology enables high-dimensional, multiplexed imaging at subcellular resolution without autofluorescence or cyclic imaging. The Hyperion™ XTi Imaging System supports three acquisition modes: Preview Mode (PM), Tissue Mode (TM), and Cell Mode (CM). PM provides rapid whole-slide scans for tissue overview within minutes, while TM captures entire tissue sections at 5-μm resolution, mapping over 40 biomarkers to reveal spatial heterogeneity. CM enables high-resolution, single-cell imaging of regions of interest (ROIs) identified in PM. Together with an automated slide loader, these modes support continuous, high-throughput tissue analysis.Methods Lung cancer tissue sections from an immunotherapy-treated patient were stained with a 34-marker IMC panel, combining the Human Immuno-Oncology IMC Panel and Maxpar IMC Cell Segmentation Kit. Imaging was performed using the Hyperion XTi Imaging System (Standard BioTools). PM was used for initial scanning, followed by automated ROI selection via Phenoplex™ software (Visiopharm®) based on three criteria: (1) tertiary lymphoid structures (TLS) expressing CD20 and CD3, (2) Granzyme B-rich regions, and (3) clusters of CD68 and Vimentin double-positive cells. Adjacent serial sections were imaged in TM for whole-slide morphological comparison.Results Tissue segmentation across all modes was performed using a deep-learning AI algorithm trained to identify key features such as vessels and TLS. CM images were analyzed at the single-cell level using iridium DNA-based segmentation. Spatial mapping of the TLS and cellular phenotyping was conducted with the Phenoplex guided workflow. Immune contexture was assessed using t-SNE plots stratified by spatial region and clinical variables.Conclusions This study demonstrates the utility of the Hyperion XTi Imaging System’s multimodal acquisition capabilities in extracting spatial and phenotypic insights from complex tissue samples. The integration of PM, TM, and CM modes with automated ROI selection and AI-driven segmentation enables efficient, high-resolution analysis of immune architecture. The Phenoplex platform facilitates rapid identification and spatial mapping of key cell populations and phenotypes, supporting advanced tissue profiling and biomarker discovery in immuno-oncology research.Ethics Approval This study was approved by the Institutional Review Board of Fukushima Medical University, approval No. 2538.