IBEX – A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

Abstract

The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only 2-6 molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a new method that overcomes major impediments to highly multi-plex tissue imaging. Iterative Bleaching Extends multi-pleXity (IBEX) uses an iterative staining and chemical bleaching method to enable high resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an ‘open’ system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and IBEX analysis of the same tissue. To facilitate data processing, we provide an open source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high content imaging to reveal the complex cellular landscape of diverse organs and tissues.Significance StatementSingle cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveal how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multi-plex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard to image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies.

Single cell spatial analysis and biomarker discovery in Hodgkin lymphoma

“…Clinical use of multiplexed IHC of up to 7 markers is becoming standardized (CLIA approved) [87][88][89][90] . Further multiplexing to 100+ marker analysis possible using cyclic imagers 61,91,92 . There are several distinctions between this pipeline and others 70,[93][94][95][96] .…”

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

“…Highly multiplexed affinity reagent-based methods allow for the detection of more than 50 protein targets in a single assay (6). The increased throughput of these approaches has provided an opportunity to develop comprehensive maps of proteins in human tissues at a rapid pace (4,8,9). However, antibody-based imaging approaches do not distinguish different proteoforms of a single protein.…”

“…These techniques employ enzymes, metals, and fluorophores as reporters to obtain highresolution maps of protein targets in tissues (5). In recent years, highly multiplexed antibody-based imaging assays such as CODEX (6,7), IBEX (8), and Cell-DIVE (9) have drastically increased the number of protein targets that can be probed in a single experiment. Alternatively, mass spectrometry (MS)-based imaging assays, including imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI), utilize antibodies labeled with rare earth metals to detect protein localization for up to ~60 protein targets at once (10,11).…”

Mapping the lymphatic system across body scales and expertise domains: A report from the 2021 National Heart, Lung, and Blood Institute workshop at the Boston Lymphatic Symposium

“…Multiplexed antibody-based imaging (Hickey et al, 2022) allows for the interrogation of tissues at single cell resolution with dozens of antibodies directed against protein biomarkers expressed by the diverse cell types and anatomical structures present in normal and diseased tissues. One such technique (Figure 4), Iterative Bleaching Extends multi-pleXity (IBEX) is a cyclic immunolabeling and chemical bleaching method that enables more than 50 parameters to be visualized in situ (Radtke et al, 2020;Radtke et al, 2022a). Importantly, advanced sequencing and imaging technologies can be integrated to provide a molecular and spatial map of human tissues as recently shown for normal and follicular lymphoma LNs (Radtke et al, 2022c).…”