Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging

Abstract

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 13C dopamine enrichment following 4–6 h of 150 μM 13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of 13C 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.

Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size

“…In the Stim samples, a correlation between vesicle size and DA concentration is found only for the LF group since Stim vesicles are smaller than Non stim vesicles (Figure 2A). It is possible that the 13 C signal is reduced by an effect of beam mixing during NanoSIMS imaging, which can take place when small sample features are sputtered together with surrounding, unlabelled material by the primary ion beam causing an underestimation of the signal at the edge of the feature [12c] …”

Chemical Imaging and Analysis of Single Nerve Cells by Secondary Ion Mass Spectrometry Imaging and Cellular Electrochemistry

“…A higher concentration of 13 C-dopamine (87.5 mM) was found in the dense core compared to the halo (16 mM). The Figures were reproduced from Jähne et al (2021) and Rabasco et al (2022) under the creative commons license.…”

“…The content of LDCVs has been evaluated by NanoSIMS imaging ( Lovrić et al, 2017 ; Thomen et al, 2020 ; Nguyen et al, 2022 ; Rabasco et al, 2022 ). Lovrić et al investigated the distribution of dopamine across LDCVs by incubating PC12 cells with 13 C-labeled L-DOPA, which is then converted to dopamine and loaded into vesicles.…”

“…The determined concentration of 13 C-dopamine in vesicles was ∼60 mM, which is in agreement with the electrochemical data ( Thomen et al, 2020 ). More recently, the absolute concentration of dopamine in each of two vesicle compartments was quantified ( Figure 4D ; Rabasco et al, 2022 ). It was found that the dense core contains a dopamine concentration of 87.5 mM, which is significantly higher compared to that in the halo (16 mM).…”

Depth Correction of 3D NanoSIMS Images Shows Intracellular Lipid and Cholesterol Distributions while Capturing the Effects of Differential Sputter Rate

“…To permit detecting a molecule of interest that lacks a distinctive elemental signature with NanoSIMS, that molecule may be labeled with a nonperturbing rare stable isotope that does not alter its native intracellular distribution or metabolism. − For example, the subcellular distributions of rare isotope-labeled cholesterol and sphingolipids have been revealed by NanoSIMS imaging. ,,− Also, drugs labeled with rare isotopes have been localized at their sites of action . Small (∼100 nm) intracellular vesicles containing rare isotope-labeled dopamine have been imaged with NanoSIMS, and a strategy was developed for quantifying the dopamine concentration within individual vesicles, providing insight into neurotransmitter release. ,− …”