Volume 2, Issue 4, Pages 225-238 (April 2016) Multiplexed Epitope-Based Tissue Imaging for Discovery and Healthcare Applications  Bernd Bodenmiller  Cell Systems  Volume 2, Issue 4, Pages 225-238 (April 2016) DOI: 10.1016/j.cels.2016.03.008 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Applications of Highly Multiplexed Epitope Imaging Approaches (A) Multiplexed imaging provides a comprehensive, spatially resolved view on cell types and their state in tissues. (B) Statistical and machine learning approaches reveal tissue motifs and enable development of spatial network models. (C) Correlation of highly multiplexed epitope imaging with clinical data supports biomarker discovery. (D) Highly multiplexed epitope imaging can be used to identify drug targets and to assess how a drug is distributed in a tissue and the effects it elicits. (E) Highly multiplexed epitope imaging could be a core tool for future precision medicine applications. Cell Systems 2016 2, 225-238DOI: (10.1016/j.cels.2016.03.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 General Workflow for Highly Parallelized Epitope Analysis in Tissues Tissues are isolated and preserved, then processed for epitope labeling and analyzed by highly multiplexed imaging in parallel with appropriate standards. The thus generated data are used to generate tissue images. After cell segmentation, downstream data analyses and visualization are performed. Cell Systems 2016 2, 225-238DOI: (10.1016/j.cels.2016.03.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Immunofluorescence versus Mass Cytometry for Highly Multiplexed Imaging (A) Serial immunofluorescence imaging uses cycles of tissue staining and imaging with one or a few fluorescently labeled antibodies, quenching of the fluorescence by fluorochrome destruction or antibody removal, and re-staining with additional antibodies. (B) In CyTOF imaging mass cytometry, a tissue is stained with dozens of metal-labeled antibodies simultaneously. A high-resolution laser ablation system is then used to transfer the tissue spot-by-spot into the CyTOF mass cytometer to determine metal isotope content and, therefore, epitope expression. (C) In multiplexed ion beam imaging, the tissue is stained with ten metal-labeled antibodies simultaneously. A primary ion beam is used to raster over the tissue to generate secondary ions, among them the metal isotopes that were bound to the antibodies. A sector field mass spectrometer is then used to determine the metal isotope content and, therefore, epitope expression in each rastered area. ICP-TOF MS, inductively coupled plasma time-of-flight mass spectrometer. Mass spectrometer, MS. Cell Systems 2016 2, 225-238DOI: (10.1016/j.cels.2016.03.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Information Content of Highly Multiplexed Images (A) Epitopes expressed in a given region of the sample of interest can be detected. (B) In combination with single-cell segmentation, complex cell phenotypes can be inferred and analyzed. (C) Exploiting spatial resolution, cell-cell interactions and cell microenvironments can be studied. Cell Systems 2016 2, 225-238DOI: (10.1016/j.cels.2016.03.008) Copyright © 2016 Elsevier Inc. Terms and Conditions