3D-IBEX: Achieving multiplex 3-dimensional imaging for deep phenotyping of cells in tissues

Abstract

Abstract 2D multiplexed antibody-based imaging provides a framework to study cell-cell interactions in thin tissue sections but lacks the ability to interrogate spatial relationships in larger 3D anatomical structures. Several methods for 3D imaging of optically cleared tissue exist, but current approaches do not provide the marker depth afforded by high content 2D imaging. To overcome this constraint, we combined a fast hydrophilic tissue clearing technique, clearing-enhanced 3D (Ce3D), with the recently developed Iterative Bleaching Extends Multiplexity (IBEX) technique, and created 3D-IBEX. 3D-IBEX offers a high-resolution multiplex 3D imaging method. It has been used to analyze murine lung, lymph nodes, retina, and cornea as well as human retina and jejunum, yielding seamless views of large-scale tissue structures while obtaining single-cell resolution of multiple markers, permitting the identification of discrete cell subsets and structures. To date, up to 15 markers have been visualized in a single sample using 3 iterative staining and imaging cycles. This represents a major advance in the emerging field of multiplexed high-resolution 3D imaging but requires further refinements to address issues such as slow antibody penetration, the need for new computational algorithms to process large datasets, and implementation of methods for optimal registration of iterative images. When fully realized, 3D-IBEX will open new avenues for characterization of rare cell subsets missed in thin tissue sections, visualization of structures like nerves and vessels whose trajectories in a tissue are disrupted by sectioning, and delineation of mesoscale tissue domains that contribute to homeostasis and are often the substrates for pathology. Intramural NIAID Research Opportunities (INRO) program.