Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging
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) imagi...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/1/160 |
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| author | Stefania Rabasco Tho D. K. Nguyen Chaoyi Gu Michael E. Kurczy Nhu T. N. Phan Andrew G. Ewing |
| author_facet | Stefania Rabasco Tho D. K. Nguyen Chaoyi Gu Michael E. Kurczy Nhu T. N. Phan Andrew G. Ewing |
| author_sort | Stefania Rabasco |
| collection | DOAJ |
| description | 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 <sup>13</sup>C dopamine enrichment following 4–6 h of 150 μM <sup>13</sup>C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of <sup>13</sup>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. |
| first_indexed | 2024-03-10T03:39:49Z |
| format | Article |
| id | doaj.art-a3e4dd3569de458e896dee30c22f19bf |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T03:39:49Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-a3e4dd3569de458e896dee30c22f19bf2023-11-23T11:35:37ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-12-0123116010.3390/ijms23010160Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS ImagingStefania Rabasco0Tho D. K. Nguyen1Chaoyi Gu2Michael E. Kurczy3Nhu T. N. Phan4Andrew G. Ewing5Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, SwedenDepartment of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, SwedenDepartment of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, SwedenDrug Metabolism and Pharmacokinetics, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, SE-431 83 Mölndal, SwedenDepartment of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, SwedenDepartment of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, SwedenThe 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 <sup>13</sup>C dopamine enrichment following 4–6 h of 150 μM <sup>13</sup>C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of <sup>13</sup>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.https://www.mdpi.com/1422-0067/23/1/160NanoSIMSdense core vesiclesvesicular compartmentalizationdopamineabsolute quantification |
| spellingShingle | Stefania Rabasco Tho D. K. Nguyen Chaoyi Gu Michael E. Kurczy Nhu T. N. Phan Andrew G. Ewing Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging International Journal of Molecular Sciences NanoSIMS dense core vesicles vesicular compartmentalization dopamine absolute quantification |
| title | Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging |
| title_full | Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging |
| title_fullStr | Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging |
| title_full_unstemmed | Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging |
| title_short | Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging |
| title_sort | localization and absolute quantification of dopamine in discrete intravesicular compartments using nanosims imaging |
| topic | NanoSIMS dense core vesicles vesicular compartmentalization dopamine absolute quantification |
| url | https://www.mdpi.com/1422-0067/23/1/160 |
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