Confocal Measurements Research Articles

Indoxyl sulfate deteriorates cardiac electrophysiological function and increases the likelihood of arrhythmias in human ventricular myocardial cells

Abstract Background Approximately 10% of the world population is suffering from chronic kidney disease (CKD). In these patients, an increased mortality and morbidity, predominantly derived from cardiovascular diseases, can be observed. Purpose The impact of CKD on myocardial cell mechanisms is only partially understood. This study aims to investigate the impact of the uremic toxin indoxyl sulfate (IS) on human ventricular myocardial cells. Methods Human left ventricular myocardium was obtained from patients with severe aortic stenosis who underwent surgical valve replacement. In addition, human ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) obtained from donors without any known cardiovascular disease were used. Before measurements were conducted, isolated single primary human cardiomyocytes were incubated for 15 minutes with an IS concentration of 120 mg/l (highest measured IS-level in dialysis patients). Further, iPSC-CM were incubated for one week with IS 45 mg/l and IS 120 mg/l, with IS 45 mg/l representing the average dialysis patients IS-level. Epifluorescence and confocal microscopy were performed to investigate Ca2+hemostasis and patch clamp measurements assessed cardiac action potentials and the occurrence of arrythmias. Results Experiments were conducted using human ventricular myocardium from 10 patients with a mean left ventricular ejection fraction of 51.5±12% and a mean glomerular filtration rate of 66.3±22.7 ml/min. Compared to the control group, higher systolic Ca2+ transient amplitudes were observed after acute incubation with IS 120 mg/l in single primary human cardiomyocytes. Measurements after 7 days of IS incubation (IS 45 mg/l and 120 mg/l) in iPSC-CM confirmed these previous results by showing higher Ca2+ transient amplitudes (Figure 1). Additionally, cells showed a diminished diastolic Ca2+ level compared to controls. An increased Ca2+ spark frequency in IS-treated iPSC-CM versus the control group was unveiled by confocal microscopy measurements. While an increased number of delayed afterdepolarizations could be observed, IS seemed to have no effect on cardiac action potential duration in iPSC-CM. Conclusion For the first time, we could reveal the impact of IS on cellular electrophysiology of primary human myocardium cells and iPSC-CM and identify IS as a proarrhythmogenic trigger. Therefore, IS may be of relevance in explaining the high cardiovascular mortality in end-stage CKD patients. To improve the outcome of these patients, new therapeutic approaches will be necessary since IS, a protein-bound uremic toxin, is only insufficiently removed by dialysis.

Capsaicinoids and cytotoxic activity in Capsicum annuum var. conoides

Two novel capsaicinoids (3, 4) , a new amide alkaloid (1), and three new natural compounds(7, 10, 12)in addition to seven known capsaicinoids (5, 6, 8, 9, 11, 13, 14) and one known amide alkaloid (2), were isolated from Capsicum annuum var. conoides. Structure of these novel compounds were elucidated by HRESIMS and 1D/2D NMR spectroscopy data. Compounds 1–14 were tested against five human cells (A549, MCF-7, HEPG2, SKOV3, L02) by means of a proliferation assay using CCK-8 to evaluate their toxicity. The results showed that compound 3 (IC50 20.50 ± 0.49 μM) had inhibitory activity against MCF-7 cells which was superior to HCPT (24.87 ± 0.91 μM). Compound 3 pairs of confocal mitochondrial potential measurements and apoptosis studies had shown that it induces dose-dependent apoptosis in MCF-7 cells. The prediction of compound 3 and breast cancer-related targets through network pharmacology and molecular docking suggests that it has the potential to be developed into an anti-tumor drug and may offer valuable insights for future therapeutic strategies.

Phase behavior and interaction of strong polyelectrolyte dextran sulfate and whey protein isolation: Effects of pH, protein/polysaccharide ratio, and salt addition

The strong polyelectrolyte dextran sulfate (DS) is an anionic polysaccharide with a high negative charge, characterized by high stability and pH independence. DS and whey protein isolate (WPI) were selected to study the specific effects of highly negatively charged polysaccharides on the phase behavior and interaction of WPI/DS complexes (1 % w/v) under varying external conditions (pH, WPI:DS ratio, and salt addition). The phase diagrams, zeta potential, and laser confocal scanning microscopy measurements indicated that the WPI/DS complexes did not dissociate even at pH 1 due to the pH independence of DS. The exclusion volume effect of DS promoted WPI self-aggregation at high salt concentrations, which inhibited acidification-induced dissociation. Isothermal titration calorimetry indicated that the WPI/DS interaction is a spontaneous exothermic reaction driven by both enthalpy and entropy changes due to electrostatic interactions. This study provides valuable information on the interactions between highly negatively charged polysaccharides and proteins.

Noninvasive Multitechnique Analysis of Baroque Amber Artworks From Gdansk: The Benefits of Using Raman Spectroscopy

ABSTRACTThe multidisciplinary, noninvasive analysis of baroque amber artworks is part of a research project that deals with the most valuable objects from the Museum of Gdansk (Poland). The two most interesting objects will be presented here: a Baroque Gdansk wardrobe‐shaped amber cabinet (made by Johann Georg Zernebach, Gdansk, 1724) and a crucifix (Gdansk, 17th century). Macro‐ (UV, IR, and X‐radiography) and micro‐ (XRF and Raman spectroscopy) analytical methods were applied to reveal traces of old conservation treatments and uncover the techniques of the 17th and 18th century amber masters. UV photography shows the differences between the amber plates that are not so easily detected under visible light, while XRF spectrometry detects elements atypical for amber objects, suggesting previous conservation treatments. Confocal Raman measurements, especially Raman depth profiling, were performed for the amber artworks and allowed to detect areas of previous conservation treatments. Single‐point scans were collected from the surface (0 μm) to a depth of −200 μm, with a step of 50 μm. The results allowed us to identify three different types of zones: where the amber was preserved without any protective layer, places where the amber was covered with a thin layer of a protective substance, and places where amber elements were compensated for loss using a binding agent and filler. The presented project allowed the development of a comprehensive methodology for the analysis of amber objects, especially to optimize the capabilities of confocal Raman microscopy. The combination of macro‐ and microanalytical techniques made it possible to obtain a broad overview of such complex artworks while optimizing the time and effort spent on the investigations.

A closer look at the effects of oxygen on the photoluminescence properties of CdSe/CdS quantum dots.

We present a detailed study on the effects of oxygen on the photoluminescence properties of CdSe/CdS quantum dots (QDs). We investigated the role of oxygen by performing confocal measurements on thin films as well as on single particles while rapidly exchanging the gaseous environment between oxygen and an inert gas atmosphere. We found that the deionization of negatively charged particles by oxygen depends on both the excitation power and the shell thickness of the QDs. For QDs with thin shells, which exhibit strong photoluminescence blinking, we observed that the presence of oxygen affects both band-edge carrier blinking and hot-carrier blinking.

Depth profiling of W[sbnd]Ta based fusion-relevant samples using picosecond laser ablation

This study presents a detailed picosecond LIBS analysis of WTa coatings on Molybdenum substrate with varying layer thicknesses for fusion relevant applications. Ps-LIBS is performed on three WTa layers; two without deuterium (∼7 μm thickness) and one with deuterium (∼1 μm thickness). The LIBS measurements are conducted under argon gas flow at 5±0.2 mbar pressure with different laser pulse energies (1 mJ, 3 mJ) and 100 spectra are recorded consecutively at one spot on the sample for different set of gate delay/gate width (200/200 ns, 300/300 ns, 450/450 ns). The obtained LIBS and Glow-Discharge Optical Emission Spectroscopy (GDOES) depth profiles are compared with the confocal microscopic measurements showing good agreement. Additionally, the ablation rate and layer thickness are calculated for different experimental conditions. The Calibration-Free LIBS approach is used for elemental analysis and the results are compared with GDOES results. The capability of ps-LIBS to quantify Ta in WTa alloy is explored for 2 at.% of Ta. However, due to higher ablation rate of laser and thin coating in WTaD sample, the layer is irradiated in few laser pulse and therefore, CF-LIBS analysis is not performed for it.

Air/water vapor control with lithium chloride/polyvinyl alcohol/sulfone composite films

With the burgeoning membrane market focusing on moisture control applications, air/water filtered membranes find increasing use in diverse fields from electronics to food preservation. Fabrication and investigations of lithium chloride (LiCl)/Polyvinyl Alcohol (PVA)/sulfone composite films designed to dehumidify the surrounding air have been conducted. These films combine hydrophilic PVA with sulfone, a material known for its water vapor selectivity. Additionally, this research analyzes the impact of LiCl concentration on the film's performance, revealing that an optimal balance between water permeability and air blockage can be achieved by adjusting material compositions. Various material characterizations, including X-ray diffraction (XRD), secondary ion mass spectrometry (SIMS), Raman spectroscopy, photoluminescence (PL) spectroscopy, transmission electron microscopy (TEM), optical microscopy (OM), and confocal optical surface measurements, shed light on the film's surface morphology, lattice structure, and optical properties. The results demonstrate that the LiCl/PVA/sulfone composite films hold promise as effective dehumidifying components with potential applications in air conditioning and smart wearable garments.

Fabrication of quantum emitters in aluminum nitride by Al-ion implantation and thermal annealing

Single-photon emitters (SPEs) within wide-bandgap materials represent an appealing platform for the development of single-photon sources operating at room temperatures. Group III-nitrides have previously been shown to host efficient SPEs, which are attributed to deep energy levels within the large bandgap of the material, in a configuration that is similar to extensively investigated color centers in diamond. Anti-bunched emission from defect centers within gallium nitride and aluminum nitride (AlN) have been recently demonstrated. While such emitters are particularly interesting due to the compatibility of III-nitrides with cleanroom processes, the nature of such defects and the optimal conditions for forming them are not fully understood. Here, we investigate Al implantation on a commercial AlN epilayer through subsequent steps of thermal annealing and confocal microscopy measurements. We observe a fluence-dependent increase in the density of the emitters, resulting in the creation of ensembles at the maximum implantation fluence. Annealing at 600 °C results in the optimal yield in SPEs formation at the maximum fluence, while a significant reduction in SPE density is observed at lower fluences. These findings suggest that the mechanism of vacancy formation plays a key role in the creation of the emitters and open enticing perspectives in the defect engineering of SPEs in solid state.

Optimization of Sustainable Production Processes in C45 Steel Machining Using a Confocal Chromatic Sensor

Metal machining production faces a myriad of demands encompassing ecology, automation, product control, and cost reduction. Within this framework, an exploration into employing a direct inspection of the machined area within the work zone of a given machine through a confocal chromatic sensor was undertaken. In the turning process, parameters including cutting speed (A), feed (B), depth of cut (C), workpiece length from clamping (D), and cutting edge radius (E) were designated as input variables. Roundness deviation (Rd) and tool face wear (KM) parameters were identified as output factors for assessing process performance. The experimental phase adhered to the Taguchi Orthogonal Array L27. Confirmatory tests revealed that optimizing process parameters according to the Taguchi method could enhance the turning performance of C45 steel. ANOVA results underscored the significant impact of cutting speed (A), feed (B), depth of cut (C), and workpiece length from clamping (D) on turning performance concerning Rd and KM. Furthermore, initial regression models were formulated to forecast roundness variation and tool face wear. The proposed parameters were found to not only influence the machined surface but also affect confocal sensor measurements. Consequently, we advocate for the adoption of these optimal cutting conditions in product production to bolster turning performance when machining C45 steel.

A metabolic map and artificial intelligence-aided identification of nasopharyngeal carcinoma via a single-cell Raman platform.

Nasopharyngeal carcinoma (NPC) is a complex cancer influenced by various factors. This study explores the use of single-cell Raman spectroscopy as a potential diagnostic tool for investigating biomolecular changes associated with NPC carcinogenesis. Seven NPC cell lines, one immortalised nasopharyngeal epithelial cell line, six nasopharyngeal mucosa tissues and seven NPC tissue samples were analysed by performing confocal Raman spectroscopic measurements and imaging. The single-cell Raman spectral dataset was used to quantify relevant biomolecules and build machine learning classification models. Metabolomic profiles were investigated using ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). By generating a metabolic map of seven NPC cell lines, we identified an interplay of altered metabolic processes involving nucleic acids, amino acids, lipids and sugars. The results from spatially resolved Raman maps and UPLC-MS/MS metabolomics were consistent, revealing an increase of unsaturated fatty acids in cancer cells, particularly in highly metastatic 5-8F and poorly differentiated CNE2 cells. The classification model achieved a nearly perfect classification when identifying NPC and non-NPC cells with an ROC-AUC of 0.99 and a value of 0.97 when identifying 13 tissue samples. This study unveils a complex interplay of metabolic network and highlights the potential roles of unsaturated fatty acids in NPC progression and metastasis. This renders further research to provide deeper insights into NPC pathogenesis, identify new metabolic targets and improve the efficacy of targeted therapies in NPC. Artificial intelligence-aided analysis of single-cell Raman spectra has achieved high accuracies in the classification of both cancer cells and patient tissues, paving the way for a simple, less invasive and accurate diagnostic test.

Polydopamine-coated photoautotrophic bacteria for improving extracellular electron transfer in living photovoltaics

Living photovoltaics are microbial electrochemical devices that use whole cell–electrode interactions to convert solar energy to electricity. The bottleneck in these technologies is the limited electron transfer between the microbe and the electrode surface. This study focuses on enhancing this transfer by engineering a polydopamine (PDA) coating on the outer membrane of the photosynthetic microbe Synechocystis sp. PCC6803. This coating provides a conductive nanoparticle shell to increase electrode adhesion and improve microbial charge extraction. A combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV–Vis absorption, and Raman spectroscopy measurements were used to characterize the nanoparticle shell under various synthesis conditions. The cell viability and activity were further assessed through oxygen evolution, growth curve, and confocal fluorescence microscopy measurements. The results show sustained cell growth and detectable PDA surface coverage under slightly alkaline conditions (pH 7.5) and at low initial dopamine (DA) concentrations (1 mM). The exoelectrogenicity of the cells prepared under these conditions was also characterized through cyclic voltammetry (CV) and chronoamperometry (CA). The measurements show a three-fold enhancement in the photocurrent at an applied bias of 0.3 V (vs. Ag/AgCl [3 M KCl]) compared to non-coated cells. This study thus lays the framework for engineering the next generation of living photovoltaics with improved performances using biosynthetic electrodes.

Measuring Partition Coefficients of In Vitro Biomolecular Condensates Using Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation is a widespread organizing principle of live cells, including, for example, the spatiotemporal organization of bacterial chromatin. The biophysics of phase-separating systems is often studied in vitro to avoid the complexity of the live-cell environment and facilitate application of advanced biophysical methods. One attractive method for measuring, e.g., partition coefficients, in such systems is fluorescence correlation spectroscopy (FCS). FCS circumvents some of the limitations of the widespread confocal laser scanning microscopy image-based measurements. Here, we describe how to perform partition coefficient measurements in biological phase-separating systems. Our protocol details typical workflows for the preparation of in vitro reconstituted condensates, FCS data acquisition, and subsequent data analysis, including corrections of some common artifacts. Our recommendations should help avoid many pitfalls of partition coefficient determination in these challenging systems.

Effects of salinity on the flow of dense colloidal suspensions.

We experimentally study the effects of salt concentration on the flowing dynamics of dense suspensions of micrometer-sized silica particles in microfluidic drums. In pure water, the particles are fully sedimented under their own weight, but do not touch each other due to their negative surface charges, which results in a "frictionless" dense colloidal suspension. When the pile is inclined above a critical angle θc ∼ 5° a fast avalanche occurs, similar to what is expected for classical athermal granular media. When inclined below this angle, the pile slowly creeps until it reaches flatness. Adding ions in solution screens the repulsive forces between particles, and the flowing properties of the suspension are modified. We observe significant changes in the fast avalanche regime: a time delay appears before the onset of the avalanche and increases with the salt concentration, the whole dynamics becomes slower, and the critical angle θc increases from ∼5° to ∼20°. In contrast, the slow creep regime does not seem to be heavily modified. These behaviors can be explained by considering an increase in both the initial packing fraction of the suspension Φ0, and the effective friction between the particles μp. These observations are confirmed by confocal microscopy measurements to estimate the initial packing fraction of the suspensions, and AFM measurements to quantify the particles surface roughness and the repulsion forces, as a function of the ionic strength of the suspensions.

Nonlinear error compensation algorithm for signal resolution of chromatic confocal measurements

The mechanisms of the nonlinear error, primarily stemming from the deviation of the polynomial model fitting the wavelength-distance mapping relationship in chromatic confocal measurement, have been elucidated. A compensation method is proposed for the nonlinear error, based on a regression tree model. Through calibration experiments using our chromatic confocal sensor, an impressive improvement of over 40 % in precision could be achieved by our proposed compensation method, which advances from ±1 μm to ±0.6 μm within a 4 mm measurement range.

Improvement of Metal-Doped β-TCP Scaffolds for Active Bone Substitutes via Ultra-Short Laser Structuring.

Various efforts have been made to develop antibacterial biomaterials capable of also sustaining bone remodulation to be used as bone substitutes and reduce patient infection rates and related costs. In this work, beta-tricalcium phosphate (β-TCP) was chosen due to its known biocompatibility and use as a bone substitute. Metal dopants were incorporated into the crystal structure of the β-TCP, and disks were produced from this material. Magnesium and strontium, as well as copper and silver, were chosen as dopants to improve the osteogenic and antibacterial properties, respectively. The surface of the β-TCP samples was further modified using a femtosecond laser system. Grid and line patterns were produced on the plates' surface via laser ablation, creating grooves with depths lower than 20 μm and widths between 20 and 40 μm. Raman and FTIR analysis confirmed that laser ablation did not result in the degradation or phase change of the materials, making it suitable for surface patterning. Laser ablation resulted in increased hydrophilicity of the materials, as the control samples (non-ablated samples) have WCA values ranging from 70° to 93° and become, upon laser ablation, superwicking surfaces. Confocal measurements show an increase in specific surface area of 50% to 200% compared to the control. Overall, the results indicate the potential of laser ablation to improve the surface characteristics of β-TCP, which may lead to an improvement in the antibacterial and osteogenic properties of the produced materials.

Modulating the Electrochemical Intercalation of Graphene Interfaces with α-RuCl3 as a Solid-State Electron Acceptor.

Intercalation reactions modify the charge density in van der Waals (vdW) materials through coupled electronic-ionic charge accumulation and are susceptible to modulation by interlayer hybridization in vdW heterostructures. Here, we demonstrate that charge transfer between graphene and α-RuCl3, which hole-dopes the graphene, greatly favors the intercalation of lithium ions into graphene-based vdW heterostructures. We systematically tune this effect on Li+ ion intercalation, modulating the intercalation potential, by using varying thicknesses of hexagonal boron nitride (hBN) as spacer layers between graphene and α-RuCl3. Confocal Raman spectroscopy and electronic transport measurements are used to monitor electrochemical intercalation, and density functional theory computations help quantify charge transfer to both α-RuCl3 and graphene upon Li intercalation. This work demonstrates a versatile approach for systematically modulating the electrochemical intercalation behavior of two-dimensional layers akin to electron donating/withdrawing substituent effects used to tune molecular redox potentials.

An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.

DNA Origami-Based Single-Molecule Force Spectroscopy and Applications.

Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion.

A hard mask process and alignment device aims to achieve high consistency and mass-scale production of gas sensors based on spraying hydrothermal gas sensing material.

The material synthesized through the hydrothermal method has received extensive and in-depth study in recent years, with a large number of literature reporting their excellent performance in the fields of catalysis or gas sensitivity. In order to combine the hydrothermal material with micro-electro-mechanical system processes to achieve large-scale manufacturing of hydrothermal synthesized materials at the wafer-level, this paper proposes a series of processes for hard mask patterned electro-atomization spraying of hydrothermal materials and designs and manufactures an alignment device that achieves the alignment of silicon hard mask and electrode wafers based on the vacuum clamping principle. Through experiments, it has been verified that this device can achieve micrometer-level alignment between the hard mask and the electrode wafer. By conducting electro-atomization spraying, hard mask patterning, optical microscopy, and confocal laser scanning microscope measurements, as well as gas sensitivity testing on a CeO2/TiO2 hydrothermal composite material published in our previous research, it was further verified that this process has good film formation consistency (Sa and Sq are both less than 3μm and the average film thickness deviation is less than 5μm), excellent and consistent gas sensitivity performance, and good long-term working stability. This article provides a promising process method for the large-scale production of hydrothermal synthesis materials at the wafer-level.

Vasculature of the Suprachiasmatic Nucleus: Pathways for Diffusible Output Signals.

Transplant studies demonstrate unequivocally that the suprachiasmatic nucleus (SCN) produces diffusible signals that can sustain circadian locomotor rhythms. There is a vascular portal pathway between the SCN and the organum vasculosum of the lamina terminalis in mouse brain. Portal pathways enable low concentrations of neurosecretions to reach specialized local targets without dilution in the systemic circulation. To explore the SCN vasculature and the capillary vessels whereby SCN neurosecretions might reach portal vessels, we investigated the blood vessels (BVs) of the core and shell SCN. The arterial supply of the SCN differs among animals, and in some animals, there are differences between the 2 sides. The rostral SCN is supplied by branches from either the superior hypophyseal artery (SHpA) or the anterior cerebral artery or the anterior communicating artery. The caudal SCN is consistently supplied by the SHpA. The rostral SCN is drained by the preoptic vein, while the caudal is drained by the basal vein, with variations in laterality of draining vessels. In addition, several key features of the core and shell SCN regions differ: Median BV diameter is significantly smaller in the shell than the core based on confocal image measurements, and a similar trend occurs in iDISCO-cleared tissue. In the cleared tissue, whole BV length density and surface area density are significantly greater in the shell than the core. Finally, capillary length density is also greater in the shell than the core. The results suggest three hypotheses: First, the distinct arterial and venous systems of the rostral and caudal SCN may contribute to the in vivo variations of metabolic and neural activities observed in SCN networks. Second, the dense capillaries of the SCN shell are well positioned to transport blood-borne signals. Finally, variations in SCN vascular supply and drainage may contribute to inter-animal differences.