Guest Editors
Andrea J. Radtke
Center for Advanced Tissue Imaging, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
Dr. Andrea Radtke obtained her PhD from Johns Hopkins University and trained as a postdoctoral fellow in the laboratory of Dr. Ronald Germain at the National Institutes of Health (NIH). In 2021, Dr. Radtke was promoted to Associate Scientist, an honorific title recognizing Staff Scientists with higher levels of expertise and scientific independence at the NIH. Dr. Radtke specializes in advanced microscopy techniques including a multiplexed antibody-based imaging method, IBEX, she co-developed. Dr. Radtke and her colleagues have used IBEX to create atlases of normal and malignant human lymph nodes as well as the human thymus. She is an Associate Member of the NIH Common Fund supported Human BioMolecular Atlas Program (HuBMAP) and a member of the Trans-NIH Lymphatics Coordinating Committee (TNLCC). Dr. Radtke is passionate about open science and co-leads several community efforts within the field of spatial biology including the IBEX Imaging Community.
Joshua Koenig
Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
Dr. Joshua Koenig obtained his PhD from McMaster University, where he is now an Assistant Professor. Dr. Koenig's research focuses on allergic immunity, in particular the responses that maintain chronic and persistent allergic conditions. Dr. Koenig was an early adopter of IBEX and is currently on the IBEX Imaging Community's steering committee. Dr. Koenig is passionate about collective and open science which supports a global research endeavour.
Collection Overview
Multiplexed antibody-based imaging facilitates the analysis of cell interactions, spatial associations, and tissue histological patterns. Despite their significant potential, several obstacles limit their widespread adoption. For these reasons, it is necessary to adopt open-source methods backed by community efforts to broaden the scope of multiplexed tissue imaging.
The Iterative Bleaching Extends Multiplexity (IBEX) is a versatile imaging method that can be completed at a relatively low-cost using instrumentation, consumables, and software available in most research settings. The IBEX protocol, along with other immunofluorescence techniques, is backed by the IBEX Imaging Community. This community comprises a global network of researchers dedicated to the open and cooperative exchange of information on multiplex imaging techniques.
To date, IBEX has been applied to many research questions, including the construction of detailed spatial atlases of mouse and human tissues, as well as the characterization of the tumor microenvironments of patient samples. Most importantly, scientific community members have expanded the original method to different application areas. The goal of this Methods Collection is to provide well-documented protocols that extend the utility of the original IBEX method by incorporating new reagents, overcoming common challenges, or demonstrating compatibility with different sample preparations and imaging configurations.
Articles
Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples
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2025
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Aleksandra Lunich1, Andrea J. Radtke2,3, Margaret Williams1, Julia M. Stern1, Daniel L. Barber4, Ronald N. Germain2, Ifeanyichukwu U. Anidi1,4
1Critical Care Medicine and Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 2Lymphocyte Biology Section and Center for Advanced Tissue Imaging, Laboratory of Immune System Biology, NIAID, NIH, 3Leica Microsystems Inc., 4T Lymphocyte Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health
Iterative Bleaching Extends Multiplicity with Use of Staining Automation for Core Facilities
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2025
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Anna E. Tseng1,2, Aoife O'Connell1, Aaron Benhamou1,3, Shivraj Yabaji1,2, Igor Kramnik1,2, Nicholas A. Crossland1,2,3, Emily Speranza4
1National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, 2Department of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine, 3Department of Virology, Immunology and Microbiology, Boston University Chobanian and Avedisian School of Medicine, 4Florida Research and Innovation Center, Cleveland Clinic Foundation
High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence
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Cited by 1
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2025
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Saven Denha*1,2, Vitoria M. Olyntho*1,2, Jake Colautti1,2, Dima Traboulsi1,2, Dan Stevens3, Susan Waserman2, Doron D. Sommer4, Manel Jordana1,2, Joshua F.E. Koenig1,2
1McMaster Immunology Research Centre, Department of Medicine, Faculty of Health Sciences, McMaster University, 2Schroeder Allergy and Immunology Research Institute, Faculty of Health Sciences, McMaster University, 3Carl Zeiss Canada MicroImaging, 4Division of Otolaryngology, Department of Surgery, McMaster University Medical Centre, McMaster University Medical Centre
Abstracts
High throughput image acquisition and analysis pipeline for evaluating skin cell phenotypes and states using IBEX and Click-iT EdU
Salome Stierli1,
Flurin Sturzenegger2,
Sofia Micheli1,
Whitney Jordaan1,
Urs Ziegler*2,
Lukas Sommer*1
1Institute of Anatomy, University of Zurich,
2Center for Microscopy and Image Analysis, University of Zurich
Optimized workflow for IBEX imaging of highly autofluorescent clinical samples
Aleksandra Lunich1,
Andrea Radtke2,
Margaret Williams1,
Julia Stern1,
Daniel Barber3,
Ronald Germain2,
Ifeanyichukwu Anidi*1
1Critical Care Medicine and Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. ,
2Lymphocyte Biology Section and Center for Advanced Tissue Imaging, Laboratory of Immune System Biology, NIAID, NIH, Bethesda, MD, USA.,
3T Lymphocyte Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA.
High-plex immunofluorescence imaging using IBEX and spectral confocal microscopy
Saven Denha1,
Vitoria Olyntho1,
Daniel Stevens2,
Doron Sommer3,
Manel Jordana1,
Joshua Koenig*1
1McMaster Immunology Research Centre, Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada.,
2Carl Zeiss Canada MicroImaging, Toronto, ON, Canada.,
3Division of Otolaryngology, Department of Surgery, McMaster University Medical Centre, McMaster University, Hamilton, ON, Canada
Where IBEX meets RNAscope: Bex-Plex
Vivien Maltez1
1University of California San Diego