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1.	Aguei-Gonzalez, P., et al. (författare) Secondary Ion Mass Spectrometry Imaging Reveals Changes in the Lipid Structure of the Plasma Membranes of Hippocampal Neurons following Drugs Affecting Neuronal Activity 2021 Ingår i: Acs Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 12:9, s. 1542-1551 Tidskriftsartikel (refereegranskat)abstract The cellular functions of lipids in the neuronal plasma membranes have been increasingly acknowledged, particularly their association to neuronal processes and synaptic plasticity. However, the knowledge of their regulatory mechanisms in neuronal cells remains sparse. To address this, we investigated the lipid organization of the plasma membranes of hippocampal neurons in relation to neuronal activity using secondary ion mass spectrometry imaging. The neurons were treated with drugs, particularly tetrodotoxin (TTX) and bicuculline (BIC), to induce chronic activation and silencing. Distinct lipid organization was found in the plasma membrane of the cell body and the neurites. Moreover, significant alterations of the levels of the membrane lipids, especially ceramides, phosphatidylserines, phosphatidic acids, and triacylglycerols, were observed under the TTX and BIC treatments. We suggest that many types of membrane lipids are affected by, and may be involved in, the regulation of neuronal function.
2.	Agui-Gonzalez, P., et al. (författare) Gold-Conjugated Nanobodies for Targeted Imaging Using High-Resolution Secondary Ion Mass Spectrometry 2021 Ingår i: Nanomaterials. - : MDPI AG. - 2079-4991. ; 11:7 Tidskriftsartikel (refereegranskat)abstract Nanoscale imaging with the ability to identify cellular organelles and protein complexes has been a highly challenging subject in the secondary ion mass spectrometry (SIMS) of biological samples. This is because only a few isotopic tags can be used successfully to target specific proteins or organelles. To address this, we generated gold nanoprobes, in which gold nanoparticles are conjugated to nanobodies. The nanoprobes were well suited for specific molecular imaging using NanoSIMS at subcellular resolution. They were demonstrated to be highly selective to different proteins of interest and sufficiently sensitive for SIMS detection. The nanoprobes offer the possibility of correlating the investigation of cellular isotopic turnover to the positions of specific proteins and organelles, thereby enabling an understanding of functional and structural relations that are currently obscure.
3.	Berglund, E Carina, et al. (författare) Oral administration of methylphenidate blocks the effect of cocaine on uptake at the Drosophila dopamine transporter. 2013 Ingår i: ACS chemical neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 4:4, s. 566-74 Tidskriftsartikel (refereegranskat)abstract Although our understanding of the actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in mammalian central nervous systems similar to cocaine, but it is thought to elicit fewer addictive and reinforcing effects owing to slower pharmacokinetics for different routes of administration between the drugs. This study utilizes the fruit fly model system to quantify the effects of oral methylphenidate on dopamine uptake during direct cocaine exposure to the fly CNS. The effect of methylphenidate on the dopamine transporter has been explored by measuring the uptake of exogenously applied dopamine. The data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter and the inhibition is concentration dependent. The peak height increased to 150% of control when cocaine was used to block the dopamine transporter for untreated flies but only to 110% for methylphenidate-treated flies. Thus, the dopamine transporter is mostly inhibited for the methylphenidate-fed flies before the addition of cocaine. The same is true for the rate of the clearance of dopamine measured by amperometry. For untreated flies the rate of clearance changes 40% when the dopamine transporter is inhibited with cocaine, and for treated flies the rate changes only 10%. The results were correlated to the in vivo concentration of methylphenidate determined by CE-MS. Our data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter for cocaine uptake, and the inhibition is concentration dependent.
4.	Berlin, Emmanuel, et al. (författare) Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging 2024 Ingår i: TALANTA. - 0039-9140 .- 1873-3573. ; 272 Tidskriftsartikel (refereegranskat)abstract Membrane lipids have been known to influence multiple signalling and cellular processes. Dysregulation of lipids at the neuronal membrane is connected to a significant alteration of the brain function and morphology, leading to brain diseases and neurodegeneration. Understanding the lipid composition and turnover of neuronal membrane will provide a significant insight into the molecular events underlying the regulatory effects of these biomolecules in a neuronal system. In this study, we aimed to characterize the composition and turnover of the plasma membrane lipids in human neural progenitor cells (NPCs) at an early differentiation stage into midbrain neurons using ToF-SIMS imaging. Lipid composition of the native plasma membrane was explored, followed by an examination of the lipid turnover using different isotopically labelled lipid precursors, including 13C-choline, 13C-lauric acid, 15N-linoleic, and 13C-stearic. Our results showed that differentiating NPCs contain a high abundance of ceramides, glycerophosphoserines, neutral glycosphingolipids, diradylglycerols, and glycerophosphocholines at the plasma membrane. In addition, different precursors were found to incorporate into different membrane lipids which are specific for the short- or long -carbon chains, and the unsaturation or saturation stage of the precursors. The lipid structure of neuronal membrane reflects the differentiation status of NPCs, and it can be altered significantly using a particular lipid precursor. Our study illustrates a potential of ToF-SIMS imaging to study native plasma membrane lipids and elucidate complex cellular processes by providing molecular -rich information at a cell level.
5.	Hanrieder, Jörg, 1980, et al. (författare) Imaging mass spectrometry in neuroscience. 2013 Ingår i: ACS chemical neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 4:5, s. 666-79 Tidskriftsartikel (refereegranskat)abstract Imaging mass spectrometry is an emerging technique of great potential for investigating the chemical architecture in biological matrices. Although the potential for studying neurobiological systems is evident, the relevance of the technique for application in neuroscience is still in its infancy. In the present Review, a principal overview of the different approaches, including matrix assisted laser desorption ionization and secondary ion mass spectrometry, is provided with particular focus on their strengths and limitations for studying different neurochemical species in situ and in vitro. The potential of the various approaches is discussed based on both fundamental and biomedical neuroscience research. This Review aims to serve as a general guide to familiarize the neuroscience community and other biomedical researchers with the technique, highlighting its great potential and suitability for comprehensive and specific chemical imaging.
6.	Hoang Philipsen, Thuy Mai, 1988, et al. (författare) Interplay between Cocaine, Drug Removal, and Methylphenidate Reversal on Phospholipid Alterations in Drosophila Brain Determined by Imaging Mass Spectrometry 2020 Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:5, s. 806-813 Tidskriftsartikel (refereegranskat)abstract Cocaine dependence displays a broad impairment in cognitive performance including attention, learning, and memory. To obtain a better understanding of the action of cocaine in the nervous system, and the relation between phospholipids and memory, we have investigated whether phospholipids recover in the brain following cocaine removal using the fly model, Drosophila melanogaster. In addition, the effects of methylphenidate, a substitute medication for cocaine dependence, on fly brain lipids after cocaine abuse are also determined to see if it can rescue the lipid changes caused by cocaine. Time of flight secondary ion mass spectrometry with a (CO2)6000+ gas cluster ion beam was used to detect intact phospholipids. We show that cocaine has persistent effects, both increasing and decreasing the levels of specific phosphatidylethanolamines and phosphatidylinositols. These changes remain after cocaine withdrawal and are not rescued by methylphenidate. Cocaine is again shown to generally increase the levels of phosphatidylcholines in the fly brain; however, after drug withdrawal, the abundance of these lipids returns to the original level and methylphenidate treatment of the flies following cocaine exposure enhances the reversal of the lipid level reducing them below the original control. The study provides insight into the molecular effects of cocaine and methylphenidate on brain lipids. We suggest that phosphatidylcholines could be a potential target for the treatment of cocaine abuse as well as be a significant hallmark of cognition and memory loss with cocaine.
7.	Hu, Keke, et al. (författare) Electrochemical Measurements Reveal Reactive Oxygen Species in Stress Granules** 2021 Ingår i: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 60:28, s. 15302-15306 Tidskriftsartikel (refereegranskat)abstract Stress granules (SGs) are membrane-less organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arsenite-induced SGs, and H2O2 is the main species. The release kinetics of H2O2 from single SGs and the number of H2O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple endpoint. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases (NDs), cancers and viral infections. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.
8.	Hu, K. K., et al. (författare) Single Exosome Amperometric Measurements Reveal Encapsulation of Chemical Messengers for Intercellular Communication 2023 Ingår i: Journal of the American Chemical Society. - 0002-7863. ; 145:21, s. 11499-11503 Tidskriftsartikel (refereegranskat)abstract In multicellular organisms, cellstypically communicateby sendingand receiving chemical signals. Chemical messengers involved in theexocytosis of neuroendocrine cells or neurons are generally assumedto only originate from the fusing of intracellular large dense corevesicles (LDCVs) or synaptic vesicles with the cellular membrane followingstimulation. Accumulated evidence suggests that exosomes oneof the main extracellular vesicles (EVs)carrying cell-dependentDNA, mRNA, proteins, etc., play an essential role in cellular communication.Due to experimental limitations, it has been difficult to monitorthe real-time release of individual exosomes; this restricts a comprehensiveunderstanding of the basic molecular mechanisms and the functionsof exosomes. In this work, we introduce amperometry with microelectrodesto capture the dynamic release of single exosomes from a single livingcell, distinguish them from other EVs, and differentiate the moleculesinside exosomes and those secreted from LDCVs. We show that, similarto many LDCVs and synaptic vesicles, exosomes released by neuroendocrinecells also contain catecholamine transmitters. This finding revealsa different mode of chemical communication via exosome-encapsulatedchemical messengers and a potential interconnection between the tworelease pathways, changing the canonical view of exocytosis of neuroendocrinecells and possibly neurons. This defines a new mechanism for chemicalcommunication at the fundamental level and opens new avenues in theresearch of the molecular biology of exosomes in the neuroendocrineand central nervous systems.
9.	Lanekoff, Ingela, 1975, et al. (författare) Mass spectrometry imaging of freeze-dried membrane phospholipids of dividing Tetrahymena pyriformis 2013 Ingår i: Surface and Interface Analysis. - : Wiley. - 0142-2421 .- 1096-9918. ; 45:1, s. 211-214 Tidskriftsartikel (refereegranskat)abstract Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze-dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze drying ex situ causes the celia to collapse and cover the plasma membrane.
10.	Lange, Felix, et al. (författare) Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging 2021 Ingår i: PLoS ONE. - 1932-6203. ; 16 Tidskriftsartikel (refereegranskat)abstract Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.
11.	Lork, A. A., et al. (författare) Chemical Imaging and Analysis of Single Nerve Cells by Secondary Ion Mass Spectrometry Imaging and Cellular Electrochemistry 2022 Ingår i: Frontiers in Synaptic Neuroscience. - : Frontiers Media SA. - 1663-3563. ; 14 Tidskriftsartikel (refereegranskat)abstract A nerve cell is a unit of neuronal communication in the nervous system and is a heterogeneous molecular structure, which is highly mediated to accommodate cellular functions. Understanding the complex regulatory mechanisms of neural communication at the single cell level requires analytical techniques with high sensitivity, specificity, and spatial resolution. Challenging technologies for chemical imaging and analysis of nerve cells will be described in this review. Secondary ion mass spectrometry (SIMS) allows for non-targeted and targeted molecular imaging of nerve cells and synapses at subcellular resolution. Cellular electrochemistry is well-suited for quantifying the amount of reactive chemicals released from living nerve cells. These techniques will also be discussed regarding multimodal imaging approaches that have recently been shown to be advantageous for the understanding of structural and functional relationships in the nervous system. This review aims to provide an insight into the strengths, limitations, and potentials of these technologies for synaptic and neuronal analyses.
12.	Lork, Alicia, et al. (författare) Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging 2024 Ingår i: CHEMICAL SCIENCE. - 2041-6520 .- 2041-6539. Tidskriftsartikel (refereegranskat)abstract Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones. Many previous studies on cellular protein turnover have used stable isotopic labelling by amino acids in cell culture (SILAC), followed by proteomic bulk analysis. However, this approach does not take into account the heterogeneity observed at the single-cell and subcellular levels. To address this, we investigated the protein turnover of neural progenitor cells at the subcellular resolution, using correlative TEM and NanoSIMS imaging, relying on a pulse-chase analysis of isotopically-labelled protein precusors. Cellular protein turnover was found significantly heterogenous across individual organelles, which indicates a possible relation between protein turnover and subcellular activity. In addition, different isotopically-labelled amino acids provided different turnover patterns, in spite of all being protein precursors, suggesting that they undergo distinct protein synthesis and metabolic pathways at the subcellular level. Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones.
13.	Meschkat, Martin, et al. (författare) White matter integrity in mice requires continuous myelin synthesis at the inner tongue 2022 Ingår i: NATURE COMMUNICATIONS. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1 Tidskriftsartikel (refereegranskat)abstract Myelin is formed of proteins of long half-lives. The mechanisms of renewal of such a stable structure are unclear. Here, the authors show that myelin integrity requires continuous myelin synthesis at the inner tongue, contributing to the maintenance of a functional axon-myelin unit. Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.
14.	Mohammadi, Amir Saeid, 1984, et al. (författare) Intact lipid imaging of mouse brain samples: MALDI, nanoparticle-laser desorption ionization, and 40 keV argon cluster secondary ion mass spectrometry 2016 Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 408:24, s. 6857-6868 Tidskriftsartikel (refereegranskat)abstract We have investigated the capability of nanoparticle-assisted laser desorption ionization mass spectrometry (NP-LDI MS), matrix-assisted laser desorption ionization (MALDI) MS, and gas cluster ion beam secondary ion mass spectrometry (GCIB SIMS) to provide maximum information available in lipid analysis and imaging of mouse brain tissue. The use of Au nanoparticles deposited as a matrix for NP-LDI MS is compared to MALDI and SIMS analysis of mouse brain tissue and allows selective detection and imaging of groups of lipid molecular ion species localizing in the white matter differently from those observed using conventional MALDI with improved imaging potential. We demonstrate that high-energy (40 keV) GCIB SIMS can act as a semi-soft ionization method to extend the useful mass range of SIMS imaging to analyze and image intact lipids in biological samples, closing the gap between conventional SIMS and MALDI techniques. The GCIB SIMS allowed the detection of more intact lipid compounds in the mouse brain compared to MALDI with regular organic matrices. The 40 keV GCIB SIMS also produced peaks observed in the NP-LDI analysis, and these peaks were strongly enhanced in intensity by exposure of the sample to trifluororacetic acid (TFA) vapor prior to analysis. These MS techniques for imaging of different types of lipids create a potential overlap and cross point that can enhance the information for imaging lipids in biological tissue sections.
15.	Nguyen, Tho D. K., et al. (författare) Quantitative Chemical Imaging at the Cellular Level: SIMS, Fluorescence, and Correlative Techniques 2022 Ingår i: Single Cell ‘Omics of Neuronal Cells. - New York, NY : Springer US. - 0893-2336. ; , s. 219-250 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.
16.	Nguyen, Tho D. K., et al. (författare) Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells 2022 Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:3, s. 4831-4842 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.
17.	Oomen, Pieter E., et al. (författare) Chemical Analysis of Single Cells 2019 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 91:1, s. 588-621 Tidskriftsartikel (refereegranskat)
18.	Phan, Nhu TN, 1981, et al. (författare) Capillary Electrophoresis-Mass Spectrometry-Based Detection of Drugs and Neurotransmitters in Drosophila Brain 2013 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 85:17, s. 8448-8454 Tidskriftsartikel (refereegranskat)abstract Capillary electrophoresis coupled to mass spectrometry has been used to determine the in vivo concentrations of the neuroactive drug, methylphenidate, and a metabolite in, the heads of the fruit fly, Drosophila melanogaster. These concentrations, evaluated at the site of action, the brain, have been correlated with orally administrated methylphenidate. D. melanogaster has a relatively simple nervous system but possesses high-order brain functions similar to humans; thus, it has been used as a common model system in biological and genetics research. Methylphenidate has been used to mediate cocaine addiction due to its lower pharmacokinetics, which results in fewer addictive and reinforcing effects than cocaine; the effects of the drug on the nervous system, however, have not been fully understood. In addition to measurements of drug concentration, the method has been used to examine drug-dose dependence on the levels of several primary biogenic amines. Higher in vivo concentration of methylphenidate is observed with increasing feeding doses up to 25 mM methylphenidate. Furthermore, administrated methylphenidate increases the drug metabolism activity and the neurotransmitter levels; however, this increase appears to saturate at a feeding dose of 20 mM. The method developed for the fruit fly provides a new tool to evaluate the concentration of administered drug at the site of action and provides information concerning the effect of methylphenidate on the nervous system.
19.	Phan, Nhu TN, 1981, et al. (författare) High-Resolution Molecular Imaging and Its Applications in Brain and Synapses 2020 Ingår i: Volume Microscopy. - New York, NY : Springer US. - 0893-2336. ; , s. 37-58 Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract The molecular organization of the brain and its synapses is highly regulated and closely related to their biological functions. In this chapter, we introduce several super-resolution imaging technologies for brain and synapses, including optical microscopy (STED, STORM), expansion microscopy, and secondary ion mass spectrometry (SIMS, NanoSIMS). Super-resolution microscopy allows for visualization of the localization and dynamics of fluorescently labeled molecules whereas mass spectrometry imaging provides information on chemical structure and molecular turnover of the brain and synapses. The general principle, pros and cons of each technology as well as experimental considerations, such as labeling and sample preparation methods, are presented. In addition, correlative optical and mass spectrometry imaging, which appears as a recent trend of brain and synaptic imaging, is also discussed together with selected relevant applications in this research area. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.
20.	Phan, Nhu TN, 1981, et al. (författare) Imaging Mass Spectrometry for Single-Cell Analysis 2014 Ingår i: Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. - : Elsevier. - 9780124095472 Bokkapitel (refereegranskat)abstract Single-cell imaging is a very important area in biological, medical, and pharmaceutical research, yet it is one of the most challenging fields for mass spectrometry. Imaging mass spectrometry (IMS), which is a powerful label-free analytical technique, has proved its capability to chemically visualize single cells with high sensitivity, chemical selectivity, and in certain cases subcellular spatial resolution. We present here an overview of the capabilities and current progress of IMS, particularly secondary ion mass spectrometry, matrix-assisted laser desorption ionization, and desorption electrospray ionization for single-cell imaging. The principles and technical developments of each technique are introduced. Critical aspects regarding single-cell imaging are addressed, especially sensitivity, spatial resolution, and sample handling. Current achievements in this field of application are also presented for both 2-D imaging and 3-D imaging. Furthermore, we address the potential contribution of IMS single-cell ‘omics’ and discuss its future development.
21.	Phan, Nhu TN, 1981, et al. (författare) Laser Desorption Ionization Mass Spectrometry Imaging of Drosophila Brain Using Matrix Sublimation versus Modification with Nanoparticles 2016 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:3, s. 1734-1741 Tidskriftsartikel (refereegranskat)abstract Laser desorption ionization mass spectrometry (LDI-MS) is used to image brain lipids in the fruit fly, Drosophila, a common invertebrate model organism in biological and neurological studies. Three different sample preparation methods, including sublimation with two common organic matrixes for matrix-assisted laser desorption ionization (MALDI) and surface assisted laser desorption ionization (SALDI) using gold nano particles, are examined for sample profiling and imaging the fly brain. Recrystallization with trifluoroacetic acid following matrix deposition in MALDI is shown to increase the incorporation of biomolecules with one matrix, resulting in more efficient ionization, but not for the other matrix. The key finding here is that the mass fragments observed for the fly brain slices with different surface modifications are significantly different. Thus, these approaches can be combined to provide complementary analysis of chemical composition, particularly for the small metabolites, diacylglycerides, phosphatidylcholines, and triacylglycerides, in the fly brain. Furthermore, imaging appears to be beneficial using modification with gold nanoparticles in place of matrix in this application showing its potential for cellular and subcellular imaging. The imaging protocol developed here with both MALDI and SALDI provides the best and most diverse lipid chemical images of the fly brain to date with LDI.
22.	Phan, Nhu TN, 1981, et al. (författare) Lipid Structural Effects of Oral Administration of Methylphenidate in Drosophila Brain by Secondary Ion Mass Spectrometry Imaging 2015 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:8, s. 4063-4071 Tidskriftsartikel (refereegranskat)abstract We use time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effects of orally administrated methylphenidate on lipids in the brain of Drosophila melanogaster (fruit fly), a major invertebrate model system in biological study and neuroscience. TOF-SIMS imaging was carried out using a recently designed high energy 40 keV Ar-4000(+) gas cluster ion gun which demonstrated improved sensitivity for intact lipids in the fly brain compared to the 40 keV C-60(+) primary ion gun. In addition, correlation of TOF-SIMS and SEM imaging on the same fly brain showed that there is specific localization that is related to biological functions of various biomolecules. Different lipids distribute in different parts of the brain, central brain, optical lobes, and proboscis, depending on the length of the carbon chain and saturation level of fatty acid (FA) branches. Furthermore, data analysis using image principal components analysis (PCA) showed that methylphenidate dramatically affected both the distribution and abundance of lipids and their derivatives, particularly fatty acids, diacylglycerides, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in the fly brains. Our approach using TOF-SIMS imaging successfully visualizes the effects of methylphenidate on the chemical structure of the fly brain.
23.	Phan, Nhu TN, 1981 (författare) Mass spectrometry based analysis of drugs, neurotransmitters and lipids in invertebrate model systems 2015 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Mass spectrometry (MS) is one of the most universal analytical techniques due to its label-free detection principle and high chemical specificity, high selectivity, and sensitivity. MS is diverse with many different types of systems to meet different analytical demands from various research areas. MS can be used for bulk analysis, in particular when coupled with a separation tools such as capillary electrophoresis or liquid chromatography, provides highly accurate qualitative and quantitative information of sample compositions. Imaging mass spectrometry (IMS), on the other hand, allows for imaging the chemical structures in intact samples with impressive spatial resolution (< micron). In this thesis, MS is used for two main objectives. First, MS is used to investigate the concentration at the site of action of methylphenidate (MPH), and its neurological effects on the nervous system of Drosophila melanogaster (fruit fly). MPH, which is a common medical drug for attention deficit hyperactivity disorder and an alternative drug to replace cocaine during the process of quitting drug abuse, has a stimulant action similar to cocaine as it also binds to the dopamine transporter protein and thereby increases the concentration of extracellular dopamine, a neurotransmitter, in the mammalian nervous system. It has recently been evidence that MPH exhibits neurological impact in long-term use; however, the details of this disorder are not fully understood. Drosophila has been chosen as a model for these studies owing to its short life cycle, prolific reproduction, and highly conserved physiological effects with humans, especially in drug addiction. The second main objective of the thesis is to develop MSI methods for biomolecular imaging of tissue samples including Drosophila brain and C. elegans. Multimodal imaging with secondary ion mass spectrometry (SIMS) and laser desorption ionization mass spectrometry (LDI MS) of the fly brain provide complementary biomolecular information of the brain structure. The molecular signature of C. elegans, one of the primary biological models used today, is very useful for studies of cellular processes and can be related to behavior of the worm. In paper I, the in vivo concentration of MPH in Drosophila brain after oral administration was determined by capillary electrophoresis mass spectrometry (CE-MS). The information was then applied to study the effects of methylphenidate treatment on the action of cocaine on dopamine uptake in vivo in Drosophila. In paper II, capillary electrophoresis mass spectrometry was extensively used for qualitative and quantitative analysis of orally administrated methylphenidate and metabolites as well as evaluation of the drug-dose dependency of neurotransmitter concentrations in the fly brain. In paper III, an imaging protocol for Drosophila brain with SIMS, including sample preparation, data treatment with image-based principle components analysis, and continuous imaging was developed. The imaging protocol was applied in paper IV to investigate lipid structural effects of MPH on Drosophila brain. The distribution and biological functions of biomolecules in the fly brain are studied using a combination of SIMS and SEM imaging. In addition, it is demonstrated that oral administration of MPH significantly alters the distribution and abundance of various brain lipids. Paper V presents a multimodal imaging approach to Drosophila brain for lipid detection using matrix assisted laser desorption ionization (MALDI). Different surface modifications, including matrix sublimation and nanoparticle deposition, show specific detectable lipids and therefore can be used in a complementary fashion to profile biological samples. In paper VI, the chemical anatomy of C. elegans is studied using SIMS imaging. Two-dimensional and three-dimensional approaches are used for worm sections and the whole worm, respectively.
24.	Phan, Nhu TN, 1981, et al. (författare) Measuring synaptic vesicles using cellular electrochemistry and nanoscale molecular imaging 2017 Ingår i: Nature Reviews Chemistry. - : Springer Science and Business Media LLC. - 2397-3358. ; 1 Tidskriftsartikel (refereegranskat)abstract The synaptic vesicle, a cellular compartment tens to hundreds of nanometres in size, is a main player in the process of exocytosis for neuronal communication. Understanding the regulatory mechanism of neurotransmission and neurological disorders requires the quantification of chemicals transmitted between cells. These challenging single vesicle measurements can be performed using analytical techniques described in this Review. In vivo amperometry at living cells can be used to quantify the amount of neurotransmitter released from a vesicle. By contrast, intracellular vesicle impact electrochemical cytometry allows the amount of molecules to be determined inside single vesicles. Although the dominant mode of exocytosis from vesicles is still under debate, several experiments point to the importance of partial release modes. Making use of fluorescent or isotopically labelled probes enables super-resolution optical and mass spectrometric imaging of molecular composition and activity of single vesicles. Correlating results from these nanoscopic techniques with those from electrochemistry has proved advantageous in understanding the relationship between vesicle structure and function. © 2017 Macmillan Publishers Limited.
25.	Phan, Nhu TN, 1981, et al. (författare) MS/MS analysis and imaging of lipids across Drosophila brain using secondary ion mass spectrometry 2017 Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 409:16, s. 3923-3932 Tidskriftsartikel (refereegranskat)abstract Lipids are abundant biomolecules performing central roles to maintain proper functioning of cells and biological bodies. Due to their highly complex composition, it is critical to obtain information of lipid structures in order to identify particular lipids which are relevant for a biological process or metabolic pathway under study. Among currently available molecular identification techniques, MS/MS in secondary ion mass spectrometry (SIMS) imaging has been of high interest in the bioanalytical community as it allows visualization of intact molecules in biological samples as well as elucidation of their chemical structures. However, there have been few applications using SIMS and MS/MS owing to instrumental challenges for this capability. We performed MS and MS/MS imaging to study the lipid structures of Drosophila brain using the J105 and 40-keV Ar-4000 (+) gas cluster ion source, with the novelty being the use of MS/MS SIMS analysis of intact lipids in the fly brain. Glycerophospholipids were identified by MS/MS profiling. MS/MS was also used to characterize diglyceride fragment ions and to identify them as triacylglyceride fragments. Moreover, MS/MS imaging offers a unique possibility for detailed elucidation of biomolecular distribution with high accuracy based on the ion images of its fragments. This is particularly useful in the presence of interferences which disturb the interpretation of biomolecular localization.
26.	Phan, Nhu TN, 1981, et al. (författare) ToF-SIMS imaging of lipids and lipid related compounds in Drosophila brain 2014 Ingår i: Surface and Interface Analysis. - : Wiley. - 1096-9918 .- 0142-2421. ; 46:S1, s. 123-126 Tidskriftsartikel (refereegranskat)abstract Drosophila melanogaster (fruit fly) has a relatively simple nervous system but possesses high order brain functions similar to humans. Therefore, it has been used as a common model system in biological studies, particularly drug addiction. Here, the spatial distribution of biomolecules in the brain of the fly was studied using time-of-flight SIMS. Fly brains were analyzed frozen to prevent molecular redistribution prior to analysis. Different molecules were found to distribute differently in the tissue, particularly the eye pigments, diacylglycerides, and phospholipids, and this is expected to be driven by their biological functions in the brain. Correlations in the localization of these molecules were also observed using principal components analysis of image data, and this was used to identify peaks for further analysis. Furthermore, consecutive analyses following 10keV Ar-2500(+) sputtering showed that different biomolecules respond differently to Ar-2500(+) sputtering. Significant changes in signal intensities between consecutive analyses were observed for high mass molecules including lipids.
27.	Philipsen, Mai H., 1988, et al. (författare) Mass Spectrometry Imaging of Cocaine, Drug Removal and Methylphenidate AlterPhospholipids in Drosophila Brain 2020 Ingår i: Biorxiv preprint. Tidskriftsartikel (refereegranskat)
28.	Philipsen, Mai H., 1988, et al. (författare) Mass Spectrometry Imaging Shows Cocaine and Methylphenidate Have Opposite Effects on Major Lipids in Drosophila Brain 2018 Ingår i: Acs Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 9:6, s. 1462-1468 Tidskriftsartikel (refereegranskat)abstract Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to study the effects of cocaine versus methylphenidate administration on both the localization and abundance of lipids in Drosophila melanogaster brain. A J105 ToF-SIMS with a 40 keV gas cluster primary ion source enabled us to probe molecular ions of biomolecules on the fly with a spatial resolution of similar to 3 mu m, giving us unique insights into the effect of these drugs on molecular lipids in the nervous system. Significant changes in phospholipid composition were observed in the central brain for both. Principal components image analysis revealed that changes occurred mainly for phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols. When the lipid changes caused by cocaine were compared with those induced by methylphenidate, it was shown that these drugs exert opposite effects on the brain lipid structure. We speculate that this might relate to the molecular mechanism of cognition and memory.
29.	Rabasco, Stefania, et al. (författare) Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging 2023 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24 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.
30.	Rabasco, Stefania, et al. (författare) Localization and absolute quantification of dopamine in discrete intravesicular compartments using nanoSIMS imaging 2022 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:1 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.
31.	Thomen, Aurélien, et al. (författare) Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles 2020 Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 14:4, s. 4316-4325 Tidskriftsartikel (refereegranskat)abstract Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.
32.	Zheng, Ying-Ning, et al. (författare) Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy. 2021 Ingår i: ACS measurement science Au. - : American Chemical Society (ACS). - 2694-250X. ; 1:3, s. 131-138 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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