Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging.

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.

Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging.

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.

Single Exosome Amperometric Measurements Reveal Encapsulation of Chemical Messengers for Intercellular Communication.

In multicellular organisms, cells typically communicate by sending and receiving chemical signals. Chemical messengers involved in the exocytosis of neuroendocrine cells or neurons are generally assumed to only originate from the fusing of intracellular large dense core vesicles (LDCVs) or synaptic vesicles with the cellular membrane following stimulation. Accumulated evidence suggests that exosomes─one of the main extracellular vesicles (EVs)─carrying cell-dependent DNA, mRNA, proteins, etc.

An iodine-containing probe as a tool for molecular detection in secondary ion mass spectrometry.

We developed here an iodine-containing probe that can be used to identify the molecules of interest in secondary ion mass spectrometry (SIMS) by simple immunolabelling procedures. The immunolabelled iodine probe was readily combined with previously-developed SIMS probes carrying fluorine, to generate dual-channel SIMS data. This probe should provide a useful complement to the currently available SIMS probes, thus expanding the scope of this technology.

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

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.

White matter integrity in mice requires continuous myelin synthesis at the inner tongue.

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.

Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells.

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 C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of C-labeled dopamine into their vesicles.

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

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 C dopamine enrichment following 4-6 h of 150 μM C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation.

Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy.

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).

Gold-Conjugated Nanobodies for Targeted Imaging Using High-Resolution Secondary Ion Mass Spectrometry.

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.

Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging.

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.

Secondary Ion Mass Spectrometry Imaging Reveals Changes in the Lipid Structure of the Plasma Membranes of Hippocampal Neurons following Drugs Affecting Neuronal Activity.

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.

Electrochemical Measurements Reveal Reactive Oxygen Species in Stress Granules*.

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 H O is the main species.

Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles.

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.

Interplay between Cocaine, Drug Removal, and Methylphenidate Reversal on Phospholipid Alterations in Brain Determined by Imaging Mass Spectrometry.

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, . 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.

Antibody-driven capture of synaptic vesicle proteins on the plasma membrane enables the analysis of their interactions with other synaptic proteins.

Many organelles from the secretory pathway fuse to the plasma membrane, to exocytose different cargoes. Their proteins are then retrieved from the plasma membrane by endocytosis, and the organelles are re-formed. It is generally unclear whether the organelle proteins colocalize when they are on the plasma membrane, or whether they disperse.

Boron-Containing Probes for Non-optical High-Resolution Imaging of Biological Samples.

Boron has been employed in materials science as a marker for imaging specific structures by electron energy loss spectroscopy (EELS) or secondary ion mass spectrometry (SIMS). It has a strong potential in biological analyses as well; however, the specific coupling of a sufficient number of boron atoms to a biological structure has proven challenging. Herein, we synthesize tags containing closo-1,2-dicarbadodecaborane, coupled to soluble peptides, which were integrated in specific proteins by click chemistry in mammalian cells and were also coupled to nanobodies for use in immunocytochemistry experiments.

Mass Spectrometry Imaging Shows Cocaine and Methylphenidate Have Opposite Effects on Major Lipids in Drosophila Brain.

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 ∼3 μm, 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.

MS/MS analysis and imaging of lipids across Drosophila brain using secondary ion mass spectrometry.

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.

Intact lipid imaging of mouse brain samples: MALDI, nanoparticle-laser desorption ionization, and 40 keV argon cluster secondary ion mass spectrometry.

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.

Laser Desorption Ionization Mass Spectrometry Imaging of Drosophila Brain Using Matrix Sublimation versus Modification with Nanoparticles.

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 nanoparticles, 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.

ToF-SIMS imaging of lipids and lipid related compounds in Drosophila brain.

(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 secondary ion mass spectrometry (ToF-SIMS).

Lipid structural effects of oral administration of methylphenidate in Drosophila brain by secondary ion mass spectrometry imaging.

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 Ar4000(+) gas cluster ion gun which demonstrated improved sensitivity for intact lipids in the fly brain compared to the 40 keV C60(+) 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.