biostudies-literatureKeren LNIBIB NIH HHSNIA NIH HHSNIDA NIH HHSNIAID NIH HHSNCI NIH HHSNINDS NIH HHSNational Institutes of HealthNIAMS NIH HHSNIH HHSNIGMS NIH HHSNational Science Foundation Graduate Research Fellowshipeaax5851https://www.ebi.ac.uk/biostudies/studies/S-EPMC6785247biostudies-literatureUnknown5(10)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 tuScience advancesMIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure.PMC6785247R01 DA022550U24 CA224309DP2 EB024246U19 AI116484U19 AI104209R01 AG057915DP5 OD019822P50 NS0626845-T32-AI07290-33R01 AG056287R01 CA183904R21 AG032494Grant No. 2017242837R01 AR067145K99 GM104148AI07290Bendall SCAngelo MAngoshtari RFienberg HBosse MWang JKeren LThompson SKambham NNolan GGreenwald NFGalli SJVijayaragavan KRisom TMarquez DWest RMcCaffrey EMontine TJKirkwood DfalseMIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure.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 tu2019-01-01T00:00:00Z2019 Oct2025-04-19T17:10:09.177Z2019-10-30T08:19:11ZS-EPMC67852473163302610.1126/sciadv.aax5851