Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue
Summary: 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 e...
| Published in: | STAR Protocols |
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| Main Authors: | , , , , , , , |
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
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| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S266616672500382X |
| _version_ | 1849376304032382976 |
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| author | Justin A. Smith Drew T. Bloss MacKenzie D. Williams Amanda L. Posgai Todd M. Brusko Mark A. Atkinson Clive H. Wasserfall Maigan A. Brusko |
| author_facet | Justin A. Smith Drew T. Bloss MacKenzie D. Williams Amanda L. Posgai Todd M. Brusko Mark A. Atkinson Clive H. Wasserfall Maigan A. Brusko |
| author_sort | Justin A. Smith |
| collection | DOAJ |
| container_title | STAR Protocols |
| description | Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-89efdbce8b014e859fd7af2f765bb31a |
| institution | Directory of Open Access Journals |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| spelling | doaj-art-89efdbce8b014e859fd7af2f765bb31a2025-08-20T03:56:41ZengElsevierSTAR Protocols2666-16672025-09-016310397610.1016/j.xpro.2025.103976Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissueJustin A. Smith0Drew T. Bloss1MacKenzie D. Williams2Amanda L. Posgai3Todd M. Brusko4Mark A. Atkinson5Clive H. Wasserfall6Maigan A. Brusko7Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USA; Corresponding authorDepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USADepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USADepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USADepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USA; Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610, USA; Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USADepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USA; Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610, USADepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USADepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Diabetes Institute, University of Florida, Gainesville, FL 32610, USA; Corresponding authorSummary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S266616672500382XImmunologyMicroscopyProtein BiochemistryProteomicsComputer sciences |
| spellingShingle | Justin A. Smith Drew T. Bloss MacKenzie D. Williams Amanda L. Posgai Todd M. Brusko Mark A. Atkinson Clive H. Wasserfall Maigan A. Brusko Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue Immunology Microscopy Protein Biochemistry Proteomics Computer sciences |
| title | Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue |
| title_full | Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue |
| title_fullStr | Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue |
| title_full_unstemmed | Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue |
| title_short | Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue |
| title_sort | protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue |
| topic | Immunology Microscopy Protein Biochemistry Proteomics Computer sciences |
| url | http://www.sciencedirect.com/science/article/pii/S266616672500382X |
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