Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial
Justin A Smith1, Drew T Bloss1, MacKenzie D Williams1
1Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USA.
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Multiplexed images of human lymphatic tissue are extensively preprocessed before cell phenotyping and spatial analysis. Here, we present KINTSUGI (knowledge integration with new technologies: simplified user-guided image processing), a protocol designed to interactively engage the user in each processing step to ensure quality control. We describe steps for parameter tuning and batch processing of raw image data including illumination correction, stitching, deconvolution, 3D-2D conversion, registration, and autofluorescence subtraction. We then detail procedures for segmentation, feature extraction, phenotyping, and spatial analysis.
