| 1. |
- Lovric, Jelena, 1980, et al.
(author)
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Correlative High-Resolution Imaging of Iron Uptake in Lung Macrophages
- 2022
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:37, s. 12798-12806
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Journal article (peer-reviewed)abstract
- Detection of iron at the subcellular level in order to gain insights into its transport, storage, and therapeutic prospects to prevent cytotoxic effects of excessive iron accumulation is still a challenge. Nanoscale magnetic sector secondary ion mass spectrometry (SIMS) is an excellent candidate for subcellular mapping of elements in cells since it provides high secondary ion collection efficiency and transmission, coupled with high-lateral-resolution capabilities enabled by nanoscale primary ion beams. In this study, we developed correlative methodologies that implement SIMS high-resolution imaging technologies to study accumulation and determine subcellular localization of iron in alveolar macrophages. We employed transmission electron microscopy (TEM) and backscattered electron (BSE) microscopy to obtain structural information and high-resolution analytical tools, NanoSIMS and helium ion microscopy-SIMS (HIM-SIMS) to trace the chemical signature of iron. Chemical information from NanoSIMS was correlated with TEM data, while high-spatial-resolution ion maps from HIM-SIMS analysis were correlated with BSE structural information of the cell. NanoSIMS revealed that iron is accumulating within mitochondria, and both NanoSIMS and HIM-SIMS showed accumulation of iron in electrolucent compartments such as vacuoles, lysosomes, and lipid droplets. This study provides insights into iron metabolism at the subcellular level and has future potential in finding therapeutics to reduce the cytotoxic effects of excessive iron loading.
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| 2. |
- Lovric, Jelena, 1980, et al.
(author)
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Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles
- 2017
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In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 11:4, s. 3446-3455
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Journal article (peer-reviewed)abstract
- We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.
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| 3. |
- Lovric, Jelena, 1980, et al.
(author)
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On the mechanism of electrochemical vesicle cytometry: chromaffin cell vesicles and liposomes
- 2016
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In: Faraday Discussions. - : Royal Society of Chemistry (RSC). - 1359-6640 .- 1364-5498. ; 193, s. 65-79
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Journal article (peer-reviewed)abstract
- The mechanism of mammalian vesicle rupture onto the surface of a polarized carbon fiber microelectrode during electrochemical vesicle cytometry is investigated. It appears that following adsorption to the surface of the polarized electrode, electroporation leads to the formation of a pore at the interface between a vesicle and the electrode and this is shown to be potential dependent. The chemical cargo is then released through this pore to be oxidized at the electrode surface. This makes it possible to quantify the contents as it restricts diffusion away from the electrode and coulometric oxidation takes place. Using a bottom up approach, lipid-only transmitter-loaded liposomes were used to mimic native vesicles and the rupture events occurred much faster in comparison with native vesicles. Liposomes with added peptide in the membrane result in rupture events with a lower duration than that of liposomes and faster in comparison to native vesicles. Diffusional models have been developed and suggest that the trend in pore size is dependent on soft nanoparticle size and diffusion of the content in the nanometer vesicle. In addition, it appears that proteins form a barrier for the membrane to reach the electrode and need to move out of the way to allow close contact and electroporation. The protein dense core in vesicles matrixes is also important in the dynamics of the events in that it significantly slows diffusion through the vesicle.
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| 4. |
- Majdi, Soodabeh, 1980, et al.
(author)
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DMSO Chemically Alters Cell Membranes to Slow Exocytosis and Increase the Fraction of Partial Transmitter Released
- 2017
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In: Chembiochem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:19, s. 1898-1902
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Journal article (peer-reviewed)abstract
- Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy; but the side effects of DMSO, especially on the cell environment, are not well understood, and controls with DMSO are not neutral at higher concentrations. Herein, electrochemical measurement techniques are applied to show that DMSO increases exocytotic neurotransmitter release, while leaving vesicular contents unchanged. In addition, the kinetics of release from DMSO-treated cells are faster than that of untreated ones. The results suggest that DMSO has a significant influence on the chemistry of the cell membrane, leading to alteration of exocytosis. A speculative chemical mechanism of the effect on the fusion pore during exocytosis is presented.
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| 5. |
- Najafinobar, Neda, 1985, et al.
(author)
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Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry
- 2016
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In: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 55:48, s. 15081-15085
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Journal article (peer-reviewed)abstract
- Electrochemical cytometry is a method developed recently to determine the content of an individual cell vesicle. The mechanism of vesicle rupture at the electrode surface involves the formation of a pore at the interface between a vesicle and the electrode through electroporation, which leads to the release and oxidation of the vesicle's chemical cargo. We have manipulated the membrane properties using excited fluorophores conjugated to lipids, which appears to make the membrane more susceptible to electroporation. We propose that by having excited fluorophores in close contact with the membrane, membrane lipids (and perhaps proteins) are oxidized upon production of reactive oxygen species, which then leads to changes in membrane properties and the formation of water defects. This is supported by experiments in which the fluorophores were placed on the lipid tail instead of the headgroup, which leads to a more rapid onset of vesicle opening. Additionally, application of DMSO to the vesicles, which increases the membrane area per lipid, and decreasing the membrane thickness result in the same enhancement in vesicle opening, which confirms the mechanism of vesicle opening with excited fluorophores in the membrane. Light-induced manipulation of membrane vesicle pore opening might be an attractive means of controlling cell activity and exocytosis. Additionally, our data confirm that in experiments in which cells or vesicle membranes are labeled for fluorescence monitoring, the properties of the excited membrane change substantially. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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| 6. |
- Dunevall, Johan, 1984, et al.
(author)
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Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision-Adsorption Events at an Electrode
- 2015
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:13, s. 4344-4346
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Journal article (peer-reviewed)abstract
- We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 mu m diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.
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| 7. |
- Lovric, Jelena, 1980, et al.
(author)
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Analysis of liposome model systems by time-of-flight secondary ion mass spectrometry
- 2014
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In: Surface and Interface Analysis: 19th International Conference on Secondary Ion Mass Spectrometry (SIMS). - : Wiley. - 0142-2421 .- 1096-9918.
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Conference paper (peer-reviewed)abstract
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is an important technique for studying chemical composition of micrometer scale objects because of its high spatial resolution imaging capabilities and chemical specificity. In this work, we focus on the application of ToF-SIMS to gain insight into the chemistry of micrometer size liposomes as a potential model for neurotransmitter vesicles. Two models of giant liposomes were analyzed: histamine and aqueous two-phase system-containing liposomes. Characterization of the internal structure of single fixed liposomes was carried out both with the Bi-3(+) and C-60(+) ion sources. The depth profiling capability of ToF-SIMS was used to investigate the liposome interior.
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| 8. |
- Lovric, Jelena, 1980, et al.
(author)
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Multimodal Imaging of Chemically Fixed Cells in Preparation for NanoSIMS
- 2016
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:17, s. 8841-8848
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Journal article (peer-reviewed)abstract
- In this work, we have employed time-of-flight secondary ion mass spectrometry (ToF-SIMS) to image chemically fixed adrenal cells prepared for transmission electron microscopy (TEM) and subsequent high-spatial-resolution NanoSIMS imaging. The sample fixation methodology preserves cell morphology, allows analysis in the ultrahigh vacuum environment, and reduces topographic artifacts, thus making these samples particularly favorable for ToF-SIMS analysis. ToF-SIMS imaging enables us to determine the chemistry and preservation capabilities of the chemical fixation as well as to locate specific ion species from OsO4. The OsO4 species have been localized in lysosomes of cortical cells, a type of adrenal cell present in the culture. NanoSIMS imaging of the 190Os16O– ion species in cortical cells reveals the same localization as a wide range of OsO4 ions shown with ToF-SIMS. Even though we did not use during NanoSIMS imaging the exact OsxOy– ion species discovered with ToF-SIMS, ToF-SIMS allowed us to define the specific subcellular features in a high spatial resolution imaging mode. This study demonstrates the possibility for application of ToF-SIMS as a screening tool to optimize high-resolution imaging with NanoSIMS, which could replace TEM for localization in ultrahigh resolution imaging analyses.
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| 9. |
- Lovric, Jelena, 1980
(author)
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PROBING SECRETORY VESICLES AND LIPOSOME MODEL SYSTEMS USING NANOSCALE ELECTROCHEMISTRY AND MASS SPECTROMETRY
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Cellular communication is based on the process of exocytosis, regulated release of chemical messengers into the extracellular space. These messengers, neurotransmitters, are packed into vesicles that during exocytosis fuse with the plasma membrane and release their content. Much work has been done to understand the mechanism of exocytosis, whether it is full or transitory transmitter release or a combination of these modes of exocytosis. However, its full comprehension is still under debate. One of the important pieces of the exocytosis puzzle and relevant evidence for the fractional exocytosis, is probing the entire neurotransmitter content of secretory vesicles. Despite the challenge to achieve this, owing to small vesicle size and often less than an attomole of detectable material, advances in bioanalytical techniques have allowed measurements at higher spatial resolution and with decreasing amounts of analytes. In this thesis, I have employed electrochemical techniques and imaging mass spectrometry as analytical tools to study the content of large dense core vesicles of neuroendocrine cells and liposome systems as secretory vesicle models.In the first part of the thesis work, Papers I and II, I applied a new amperometric technique called vesicle electrochemical cytometry to measure the catecholamine content in native vesicles and liposomes. In Paper I, this technique was employed to quantify the content of catecholamines present in single mammalian vesicles isolated from cells of the bovine adrenal gland. Paper II represents the continuation of work done in Paper I, where the same experimental setup was employed with focus on investigation of the mechanism of vesicle rupture onto the electrode by applying a bottom up approach and probing the transmitter loaded liposomes. The second part of this thesis, Papers III, IV and V, describe the application of imaging mass spectrometry to reveal the chemical composition of vesicles and liposomes. In Paper III, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to study the chemistry of micrometer size liposome models containing histamine and liposomes containing an aqueous two-phase system, both to mimic secretory vesicles. Paper IV demonstrates the potential of ToF-SIMS to evaluate the preservation capabilities of chemical fixation, a common approach for sample preparation in subcellular imaging as well as a screening tool for optimization of high-resolution NanoSIMS imaging. In Paper V, NanoSIMS, and electrochemical techniques were used in combination, to study the neurochemistry of large dense core vesicles from PC12 cells. The major goals were to investigate the impact of pharmaceuticals like L-3,4-dihydroxyphenylalanine and reserpine on metabolic pathways of neurotransmitter dopamine and to quantify dopamine content in PC12 vesicles, one vesicle at a time and in subvesicular regions.
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| 10. |
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