From Insulin Measurement to Partial Exocytosis Model: Advances in Single Pancreatic Beta Cell Amperometry over Four Decades.

Single cell Amperometry (SCA) is a powerful, sensitive, high temporal resolution electrochemical technique used to quantify secreted molecular messengers from individual cells and vesicles. This technique has been extensively applied to study the process of exocytosis, and it has also been applied, albeit less frequently, to investigate insulin exocytosis from single pancreatic beta cells. Insufficient insulin release can lead to diabetes, a chronic lifestyle disorder that affects millions of people worldwide.

High-throughput optimisations for 3D chemical imaging of pharmaceutical solid oral dosage forms.

Chemical imaging of pharmaceutical solid oral dosage forms is a key technique for quality assurance and issue diagnosis. This technique can be further augmented using 3D chemical imaging serial sections and image stacking. However, the additional collection time this entails means that 3D imaging is utilised for a very niche set of applications.

Spike by spike frequency analysis of amperometry traces provides statistical validation of observations in the time domain.

Amperometry is a commonly used electrochemical method for studying the process of exocytosis in real-time. Given the high precision of recording that amperometry procedures offer, the volume of data generated can span over several hundreds of megabytes to a few gigabytes and therefore necessitates systematic and reproducible methods for analysis. Though the spike characteristics of amperometry traces in the time domain hold information about the dynamics of exocytosis, these biochemical signals are, more often than not, characterized by time-varying signal properties.

Subsets of Nanometer Vesicles in the Fly Release Differential Fractions of Vesicular Serotonin Content during Exocytosis.

Serotonin, a monoamine neurotransmitter, is important in both the central nervous system (CNS) and the peripheral nervous system. Malfunction of serotonin signaling leads to various disorders. We studied serotonin signaling from serotonergic neurons inside the ventral nerve cord of Drosophila melanogaster.

Electrochemical Monitoring of Real-Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette.

The microtubule-associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse-mimicking nanopipette to monitor vesicle dynamics induced by tau.

Emerging Data Processing Methods for Single-Entity Electrochemistry.

Single-entity electrochemistry is a powerful tool that enables the study of electrochemical processes at interfaces and provides insights into the intrinsic chemical and structural heterogeneities of individual entities. Signal processing is a critical aspect of single-entity electrochemical measurements and can be used for data recognition, classification, and interpretation. In this review, we summarize the recent five-year advances in signal processing techniques for single-entity electrochemistry and highlight their importance in obtaining high-quality data and extracting effective features from electrochemical signals, which are generally applicable in single-entity electrochemistry.

Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles.

An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed.

Omega-3 and -6 Fatty Acids Alter the Membrane Lipid Composition and Vesicle Size to Regulate Exocytosis and Storage of Catecholamines.

The two essential fatty acids, alpha-linolenic acid and linoleic acid, and the higher unsaturated fatty acids synthesized from them are critical for the development and maintenance of normal brain functions. Deficiencies of these fatty acids have been shown to cause damage to the neuronal development, cognition, and locomotor function. We combined electrochemistry and imaging techniques to examine the effects of the two essential fatty acids on catecholamine release dynamics and the vesicle content as well as on the cell membrane phospholipid composition to understand how they impact exocytosis and by extension neurotransmission at the single-cell level.

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.

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

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-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.

Hofmeister Series: From Aqueous Solution of Biomolecules to Single Cells and Nanovesicles.

Hofmeister effects play a critical role in numerous physicochemical and biological phenomena, including the solubility and/or accumulation of proteins, the activities of enzymes, ion transport in biochannels, the structure of lipid bilayers, and the dynamics of vesicle opening and exocytosis. This minireview focuses on how ionic specificity affects the physicochemical properties of biomolecules to regulate cellular exocytosis, vesicular content, and nanovesicle opening. We summarize recent progress in further understanding Hofmeister effects on biomacromolecules and their applications in biological systems.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

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.

Correlative Cellular Mass Spectrometry Imaging and Amperometry Show Dose Dependent Changes in Lipid Composition and Exocytosis.

Aberrant functioning of the proteasome has been associated with crucial pathologic conditions including neurodegeneration. Yet, the complex underlying causes at the cellular level remain unclear and there are conflicting reports of neuroprotective to neurodegenerative effects of proteasomal inhibitors such as lactacystin that are utilised as models for neurodegenerative diseases. The conflicting results may be associated with different dose regimes of lactacystin and hence we have performed a dose dependent study of the effects of lactacystin to identify concurrent changes in the cell membrane lipid profile and the dynamics of exocytosis using a combination of surface sensitive mass spectrometry and single cell amperometry.

The dynamic nature of exocytosis from large secretory vesicles. A view from electrochemistry and imaging.

In this brief review, we discuss the factors that modulate the quantum size and the kinetics of exocytosis. We also discuss the determinants which motivate the type of exocytosis from the so-called kiss-and-run to full fusion and along the intermediate mode of partial release. Kiss-and-run release comprises the transient opening of a nanometer (approx.

Advances in nano/microscale electrochemical sensors and biosensors for analysis of single vesicles, a key nanoscale organelle in cellular communication.

The study of subcellular targets and biochemical processes within a living cell is valuable for biological and medical research. Secretory vesicles, one such important intracellular target, are nanoscale lipid structures that are capable of storage, transport, and secretion of, for example, neurotransmitters, hormones, proteins or waste products. Vesicles play an essential role in intercellular communication systems, as they facilitate the release of chemical messaging agents.

Vesicle Collision Protocols for the Study of Quantum Size and Exocytotic Fraction Released.

We review the methods of vesicle impact electrochemical cytometry, intracellular impact electrochemical cytometry, and single cell amperometry and their application to measuring the storage of neurotransmitters in cellular vesicles. We provide protocols to measure vesicle content, the release of catecholamines, and from there the fraction of transmitter released in each exocytosis event. The focus here has been a combination of methods to evaluate factors related to neuronal function at the cellular level and implications in, for example, cognition.

Direct Acquisition of the Gap Height of Biological Tissue-Electronic Chemical Sensor Interfaces.

Interfacing biological tissues with electronic sensors offers the exciting opportunity to accurately investigate multiple biological processes. Accurate signal collection and application are the foundation of these measurements, but a long-term issue is the signal distortion resulting from the interface gap. The height of the gap is the key characteristic needed to evaluate or model the distortion, but it is difficult to measure.

Correction: Evaluation of science advice during the COVID-19 pandemic in Sweden.

[This corrects the article DOI: 10.1057/s41599-022-01097-5.].

Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS.

Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity.

Dysfunction of vesicular storage in young-onset Parkinson's patient-derived dopaminergic neurons and organoids revealed by single cell electrochemical cytometry.

Electrochemical cytometry based on nano-tip microelectrodes was used to quantify the vesicular storage at the single-cell level in human neurons and midbrain organoids which acted as disease models of young-onset Parkinson's disease (YOPD). Human dopaminergic (DA) neurons and midbrain organoids were derived from an induced pluripotent stem cell line from one YOPD patient. We show a significant deficiency in vesicular catecholamine storage and a slower pore forming process on the surface of the microelectrode in the DA neurons derived from the YOPD patient.

Pore-Opening Dynamics of Single Nanometer Biovesicles at an Electrified Interface.

Release from nanobiovesicles via a pore generated by membrane electroporation at an electrified interface can be monitored by vesicle impact electrochemical cytometry (VIEC) and provides rich information about the various vesicular content transfer processes, including content homeostasis, intraphase content transfer, or the transient fusion of vesicles. These processes are primarily influenced by the vesicular pore-opening dynamics at the electrified interface which has not been disclosed at the single nanobiovesicle level yet. In this work, after simultaneously measuring the size and release dynamics of individual vesicles, we employed a moving mesh-finite element simulation algorithm to reconstruct the accurate pore-opening dynamics of individual vesicles with different sizes during VIEC.

Single-Vesicle Electrochemistry Following Repetitive Stimulation Reveals a Mechanism for Plasticity Changes with Iron Deficiency.

Deficiency of iron, the most abundant transition metal in the brain and important for neuronal activity, is known to affect synaptic plasticity, causing learning and memory deficits. How iron deficiency impacts plasticity by altering neurotransmission at the cellular level is not fully understood. We used electrochemical methods to study the effect of iron deficiency on plasticity with repetitive stimulation.