| Autor: |
Rabasco S; Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden., Nguyen TDK; Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden., Gu C; Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden., Kurczy ME; Drug Metabolism and Pharmacokinetics, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83 Mölndal, Sweden., Phan NTN; Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden., Ewing AG; Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden. |
| Abstrakt: |
The absolute concentration and the compartmentalization of analytes in cells and organelles are crucial parameters in the development of drugs and drug delivery systems, as well as in the fundamental understanding of many cellular processes. Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging is a powerful technique which allows subcellular localization of chemical species with high spatial and mass resolution, and high sensitivity. In this study, we combined NanoSIMS imaging with spatial oversampling with transmission electron microscopy (TEM) imaging to discern the compartments (dense core and halo) of large dense core vesicles in a model cell line used to study exocytosis, and to localize 13 C dopamine enrichment following 4-6 h of 150 μM 13 C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of 13 C dopamine in distinct vesicle domains as well as in entire single vesicles were quantified and validated by comparison to electrochemical data. We found concentrations of 87.5 mM, 16.0 mM and 39.5 mM for the dense core, halo and the whole vesicle, respectively. This approach adds to the potential of using combined TEM and NanoSIMS imaging to perform absolute quantification and directly measure the individual contents of nanometer-scale organelles. |
