Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions. To gain spatial information, we have coupled immunohistochemical and immunocytochemical methods with high-resolution laser ablation to CyTOF mass cytometry. This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution; with the availability of additional isotopes, measurement of over 100 markers will be possible. We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell-cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry complements existing imaging approaches. It will enable basic studies of tissue heterogeneity and function and support the transition of medicine toward individualized molecularly targeted diagnosis and therapies.

In recent years, chemical imaging was prognosticated to become one of the key analytical applications for laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). However, moderate spatial resolution and the associated measurement time required for a larger sampling area, have restricted this versatile, high sensitivity technique from being routinely used in two-dimensional chemical imaging. This work describes the development and investigation of a low dispersion sample chamber (tube cell), which allows improvement of the imaging capabilities by reduction of the single LA shot duration to 30 ms (full width at 1% maximum). The new tube cell is based on a constant laminar flow and a well-controlled delivery of the laser-ablated aerosol into the transport system, leading to minimized tailing of the aerosol washout and helping to separate the signals even at repetition rates as high as 20-30 Hz. To demonstrate the improved imaging capabilities, microstructured metallic thin film patterns were analyzed at a spatial resolution of a few micrometers. The LA-ICP-MS results obtained were comparable to Synchrotron-based micro-X-ray fluorescence (SR-microXRF). The suitability of the newly designed cell for multielement acquisitions was demonstrated using a simultaneous ICP-Mattauch-Herzog-MS. Finally, the novel laser ablation cell was applied to image the distribution of a metal-tagged biomarker in a thin section of breast cancer tissue. This application demonstrates that the technique is able to produce subcellular (~1 μm) spatial resolution, which is crucial for morphological assessment in cancer diagnostics.

The combination of mass cytometry and immunohistochemistry (IHC) enables new histopathological imaging methods in which dozens of proteins and protein modifications can be visualized simultaneously in a single tissue section. The power of multiplexing combined with spatial information and quantification was recently illustrated on breast cancer tissue and was described as next-generation IHC. Robust, accurate, and high-throughput cell segmentation is crucial for the analysis of this new generation of IHC data. To this end, we propose a watershed-based cell segmentation, which uses a nuclear marker and multiple membrane markers, the latter automatically selected based on their correlation. In comparison with the state-of-the-art segmentation pipelines, which are only using a single marker for object detection, we could show that the use of multiple markers can significantly increase the segmentation power, and thus, multiplexed information should be used and not ignored during the segmentation. Furthermore, we provide a novel, user-friendly open-source toolbox for the automatic segmentation of multiplexed histopathological images.