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| 2. |
- Nguyen, Tho D. K., et al.
(författare)
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Quantitative Chemical Imaging at the Cellular Level: SIMS, Fluorescence, and Correlative Techniques
- 2022
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Ingår i: Single Cell ‘Omics of Neuronal Cells. - New York, NY : Springer US. - 0893-2336. ; , s. 219-250
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Bokkapitel (refereegranskat)abstract
- The cell is a heterogeneous chemical structure designed to accommodate its complex cellular functions in a living organism. Quantitative chemical imaging at the cellular level enables the investigation of the structural and functional molecular relation underlying cellular processes. We describe here the detailed methodology of the state-of-the-art secondary ion mass spectrometry (SIMS, NanoSIMS) and fluorescence microscopy (confocal, STED), along with selected examples for quantitative imaging at the cellular level. Correlative imaging that combines different imaging techniques is also demonstrated for selected applications in cell imaging. This chapter serves as a guideline assisting readers from unfamiliar fields of research to obtain reliable imaging at the cellular level while highlighting the strengths, limitations, and potentials of these technologies for cell imaging.
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| 3. |
- Nguyen, Tho D. K.
(författare)
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Quantitative chemical imaging to study content release from single nanovesicles
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular communication is vital for the survival of multicellular organisms. This process often relies on a highly regulated mechanism called exocytosis, which involves the release of chemical signals. During exocytosis, vesicles can fully or partially release their contents. The quantity of neurotransmitters expelled in different modes of release may have diverse effects on cellular communication by allowing cells to control the level of outgoing signals. Despite significant discoveries in understanding the components of exocytosis, there is still much to uncover regarding its regulation and implications. This thesis aims to gain a better understanding of vesicular content release, particularly in the context of partial release, using quantitative chemical imaging. This includes the integration of mass spectrometry imaging (MSI) with electron microscopy or electrochemical analysis alongside light microscopy. By doing so, a comprehensive approach can be employed to gain insights into the mechanism of vesicular content release and quantify the fraction of release. Electrochemical techniques offer the advantage of high temporal resolution and enable quantification of both stored and released molecules from vesicles. Coupled with imaging methods such as fluorescence, electron microscopy, and mass spectrometry imaging, comprehensive spatial information and chemical information can be obtained to complement the data provided by electrochemical techniques. In particular, nanoscale secondary ion mass spectrometry (NanoSIMS), a high-resolution MSI method that is capable of absolute quantification at subcellular level, was primarily used throughout this thesis. PC12 cells treated with isotopically labeled L-DOPA were examined, and NanoSIMS imaging was correlated with transmission electron microscopy (TEM) to detect and quantify the labeled dopamine in the halo and dense core compartments of large dense core vesicles (LDCVs) in paper I. Paper II introduced a dual-label approach to visualize and quantify vesicles undergoing partial release in PC12 cells by exposing them to a second label during exocytosis. Expanding on the dual-label approach, paper III investigated the influence of vesicle size on the dynamics of partial release. Furthermore, the combination of vesicle impact electrochemical cytometry (VIEC) with live fluorescence imaging was developed in paper IV allowing real-time analysis of vesicular content release from isolated labeled bovine chromaffin vesicles. Overall, these studies demonstrate the application of quantitative chemical imaging in understanding the mechanism and quantifying the fraction of release in vesicular content release.
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| 4. |
- Nguyen, Tho D. K., et al.
(författare)
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Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size
- 2023
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Ingår i: Angewandte Chemie. - 0044-8249 .- 1521-3757. ; 62:28
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Tidskriftsartikel (refereegranskat)abstract
- We used correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to quantify the contents of subvesicular compartments, and to measure the partial release fraction of C-13-dopamine in cellular nanovesicles as a function of size. Three modes of exocytosis comprise full release, kiss-and-run, and partial release. The latter has been subject to scientific debate, despite a growing amount of supporting literature. We tailored culturing procedures to alter vesicle size and definitively show no size correlation with the fraction of partial release. In NanoSIMS images, vesicle content was indicated by the presence of isotopic dopamine, while vesicles which underwent partial release were identified by the presence of an I-127-labelled drug, to which they were exposed during exocytosis allowing entry into the open vesicle prior to its closing again. Demonstration of similar partial release fractions indicates that this mode of exocytosis is predominant across a wide range of vesicle sizes.
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| 5. |
- Nguyen, Tho D. K., et al.
(författare)
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Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells
- 2022
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Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:3, s. 4831-4842
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Tidskriftsartikel (refereegranskat)abstract
- For decades, "all-or-none"and "kiss-and-run"were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically. © 2022 The Authors. Published by American Chemical Society.
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| 6. |
- Rabasco, Stefania, et al.
(författare)
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Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24
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Tidskriftsartikel (refereegranskat)abstract
- Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.
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| 7. |
- Rabasco, Stefania, et al.
(författare)
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Localization and absolute quantification of dopamine in discrete intravesicular compartments using nanoSIMS imaging
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:1
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Tidskriftsartikel (refereegranskat)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 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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| 8. |
- Zheng, Ying-Ning, et al.
(författare)
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Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy.
- 2021
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Ingår i: ACS measurement science Au. - : American Chemical Society (ACS). - 2694-250X. ; 1:3, s. 131-138
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Tidskriftsartikel (refereegranskat)abstract
- In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 μm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis.
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