MIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure

Abstract

Understanding tissue structure and function requires tools that quantify the expression of multiple proteins while preserving spatial information. Here, we describe MIBI-TOF (multiplexed ion beam imaging by time of flight), an instrument that uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution in clinical tissue sections. We demonstrate quantitative, full periodic table coverage across a five-log dynamic range, imaging 36 labeled antibodies simultaneously with histochemical stains and endogenous elements. We image fields of view up to 800 μm × 800 μm at resolutions down to 260 nm with sensitivities approaching single-molecule detection. We leverage these properties to interrogate intrapatient heterogeneity in tumor organization in triple-negative breast cancer, revealing regional variability in tumor cell phenotypes in contrast to a structured immune response. Given its versatility and sample back-compatibility, MIBI-TOF is positioned to leverage existing annotated, archival tissue cohorts to explore emerging questions in cancer, immunology, and neurobiology.

Deep Learning-Based Point-Scanning Super-Resolution Imaging

“…All point scanning imaging systems have a direct relationship between pixel resolution and imaging time, sample damage, SNR, and structural detail. Thus, the ability to use deep learning to super-sample under-sampled images provides an opportunity that extends to other point scanning systems, for example ion-based imaging systems 26,27 or high resolution cryoSTEM 28 .…”

Multiplexed Single-cell Metabolic Profiles Organize the Spectrum of Cytotoxic Human T Cells

“…In addition to lineage intrinsic factors, cell activation and tissue of residence, a cell's metabolism is influenced by its location within a specific tissue microenvironment and its interactions with neighboring cells 3,28 , together critically shaping immune responses against cancer 26,27 . The antibody-based nature of scMEP enabled us to directly transfer this approach to the recently-developed multiplexed ion beam imaging (MIBI-TOF) platform 32 , thus allowing multimodal, high-dimensional, image-based analysis of the cellular metabolic regulome, phenotypic state, and importantly, cellular interactions and localizations within the tissue microenvironment. In MIBI-TOF, a tissue section (e.g.…”

“…Quantitative imaging was performed using a custom designed MIBI-TOF mass spectrometer (IONpath), as previously described 32,76 , with an image size of 400 µm 2 and 1024 x 1024 pixels. The entire cohort of 58 FOVs was acquired over a 24 h period of continuous imaging, yielding a total of 2088 images.…”

“…To address this need, we have developed a novel approach, termed single-cell metabolic profiling (scMEP), that enables quantification of single-cell metabolic states by capturing the composition of the metabolic regulome using antibody-based proteomic platforms such as mass cytometry 31 and multiplexed ion beam imaging by time-of-flight (MIBI-TOF) 32 . These technologies make use of heavy metal-conjugated antibodies that are quantified by TOF MS, thus allowing highly multiplexed, single-cell and imaging assays 33 .…”

Metabolic Analysis at the Nanoscale with Multi‐Isotope Imaging Mass Spectrometry (MIMS)

“…Similarly, drugs or probes containing transition metals such as 47 Ag, 79 Au, Fe oxides, or Cu oxides can be detected, as can the lanthanide series of 15 elements ( 57 La through 71 Lu) (reviewed in Nuñez, Renslow, Cliff, & Anderton, 2017). The utility of the lanthanide series for multiplexed immunolabeling was established using MIMS (with an oxygen source to produce positive ions) and more recently has formed the basis of commercialized TOF-SIMS instruments that can detect >30 antibodies in parallel (Angelo et al, 2014;Keren et al, 2019).…”