Abstract
Incorporation of a stable-isotope metabolic tracer into cells or tissue can be followed at submicron resolution by multi-isotope imaging mass spectrometry (MIMS), a form of imaging secondary ion microscopy optimized for accurate isotope ratio measurement from microvolumes of sample (as small as ∼30 nm across). In a metabolic MIMS experiment, a cell or animal is metabolically labeled with a tracer containing a stable isotope. Relative accumulation of the heavy isotope in the fixed sample is then measured as an increase over its natural abundance by MIMS. MIMS has been used to measure protein turnover in single organelles, track cellular division in vivo, visualize sphingolipid rafts on the plasma membrane, and measure dopamine incorporation into dense-core vesicles, among other biological applications. In this article, we introduce metabolic analysis using NanoSIMS by focusing on two specific applications: quantifying protein turnover in single organelles of cultured cells and tracking cell replication in mouse tissues in vivo. These examples illustrate the versatility of metabolic analysis with MIMS. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Metabolic labeling for MIMS Basic Protocol 2: Embedding of samples for correlative transmission electron microscopy and MIMS with a genetically encoded reporter Alternate Protocol: Embedding of samples for correlative light microscopy and MIMS Support Protocol: Preparation of silicon wafers as sample supports for MIMS Basic Protocol 3: Analysis of MIMS data.