TY - JOUR
T1 - Localization and absolute quantification of dopamine in discrete intravesicular compartments using nanoSIMS imaging
AU - Rabasco, Stefania
AU - Nguyen, Tho D.K.
AU - Gu, Chaoyi
AU - Kurczy, Michael E.
AU - Phan, Nhu T.N.
AU - Ewing, Andrew G.
N1 - Funding Information:
Funding: This research was funded by the Knut and Alice Wallenberg Foundation, the Swedish Research Council (VR Grant No. 2017-04366), and the European Research Council (ERC Advanced Grant Project No. 787534 NanoBioNext) to A.G.E., and the Swedish Research Council (VR 2020-00815), and the Hasselblad Foundation to N.T.N.P.
Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2022/1/1
Y1 - 2022/1/1
N2 - 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 localize13C dopamine enrichment following 4–6 h of 150 μM13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of13C 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.
AB - 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 localize13C dopamine enrichment following 4–6 h of 150 μM13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of13C 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.
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U2 - 10.3390/ijms23010160
DO - 10.3390/ijms23010160
M3 - Article
C2 - 35008583
AN - SCOPUS:85121568596
SN - 1661-6596
VL - 23
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 1
M1 - 160
ER -