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Background: Biomolecular condensation via liquid-liquid phase separation (LLPS) is crucial for orchestrating cellular activities temporospatially. Although the rheological heterogeneity of biocondensates and the structural dynamics of their constituents carry critical functional information, methods to quantitatively study biocondensates are lacking. Single-molecule fluorescence research can offer insights into biocondensation mechanisms. Unfortunately, as dense condensates tend to sink inside their dilute aqueous surroundings, studying their properties via methods relying on Brownian diffusion may fail., Methods: We take a first step towards single-molecule research on condensates of Tau protein under flow in a microfluidic channel of an in-house developed microfluidic chip. Fluorescence correlation spectroscopy (FCS), a well-known technique to collect molecular characteristics within a sample, was employed with a newly commercialised technology, where FCS is performed on an array detector (AD-FCS), providing detailed diffusion and flow information., Results: The AD-FCS technology allowed characterising our microfluidic chip, revealing 3D flow profiles. Subsequently, AD-FCS allowed mapping the flow of Tau condensates while measuring their burst durations through the stationary laser. Lastly, AD-FCS allowed obtaining flow velocity and burst duration data, the latter of which was used to estimate the condensate size distribution within LLPS samples., Conclusion: Studying biocondensates under flow through AD-FCS is promising for single-molecule experiments. In addition, AD-FCS shows its ability to estimate the size distribution in condensate samples in a convenient manner, prompting a new way of investigating biocondensate phase diagrams., General Significance: We show that AD-FCS is a valuable tool for advancing research on understanding and characterising LLPS properties of biocondensates., Competing Interests: Declaration of competing interest Jelle Hendrix reports financial support was provided by Research Foundation Flanders. Stijn Dilissen reports financial support was provided by Research Foundation Flanders. Tom Kache reports financial support was provided by Research Foundation Flanders. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A technique to measure the band gap of dielectric materials with high refractive index by means of energy electron loss spectroscopy (EELS) is presented. The technique relies on the use of a circular (Bessel) aperture and suppresses Cherenkov losses and surface-guided light modes by enforcing a momentum transfer selection. The technique also strongly suppresses the elastic zero loss peak, making the acquisition, interpretation and signal to noise ratio of low loss spectra considerably better, especially for excitations in the first few eV of the EELS spectrum. Simulations of the low loss inelastic electron scattering probabilities demonstrate the beneficial influence of the Bessel aperture in this setup even for high accelerating voltages. The importance of selecting the optimal experimental convergence and collection angles is highlighted. The effect of the created off-axis acquisition conditions on the selection of the transitions from valence to conduction bands is discussed in detail on a simplified isotropic two band model. This opens the opportunity for deliberately selecting certain transitions by carefully tuning the microscope parameters. The suggested approach is experimentally demonstrated and provides good signal to noise ratio and interpretable band gap signals on reference samples of diamond, GaN and AlN while offering spatial resolution in the nm range., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We introduce an innovative specimen preparation method employing the selectivity of a wet-chemical etching step to improve data quality and success rates in the atom probe analysis of contemporary semiconductor devices. Firstly, on the example of an SiGe fin embedded in SiO 2 we demonstrate how the selective removal of SiO 2 from the final APT specimen significantly improves accuracy and reliability of the reconstructed data. With the oxide removal, we eliminate the origin of shape artefacts, i.e. the formation of a non-hemispherical tip shape, that are typically observed in the reconstructed volume of complex systems. Secondly, using the same approach, we increase success rates to ∼90% for the damage-free, 3D site-specific localization of short (250 nm), vertical Si nanowires at the specimen apex. The impact of the abrupt emitter radius change that is introduced by this specimen preparation method is evaluated as being minor using field evaporation simulation and comparison of different reconstruction schemes. The Ge content within the SiGe fin as well as the 3D boron distribution in the Si NW as resolved by atom probe analysis are in good agreement with TEM/EDS and ToF-SIMS analysis, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We present atom probe analysis of 40nm wide SiGe fins embedded in SiO 2 and discuss the root cause of artefacts observed in the reconstructed data. Additionally, we propose a simple data treatment routine, relying on complementary transmission electron microscopy analysis, to improve compositional analysis of the embedded SiGe fins. Using field evaporation simulations, we show that for high oxide to fin width ratios the difference in evaporation field thresholds between SiGe and SiO 2 results in a non-hemispherical emitter shape with a negative curvature in the direction across, but not along the fin. This peculiar emitter shape leads to severe local variations in radius and hence in magnification across the emitter apex causing ion trajectory aberrations and crossings. As shown by our experiments and simulations, this translates into unrealistic variations in the detected atom densities and faulty dimensions in the reconstructed volume, with the width of the fin being up to six-fold compressed. Rectification of the faulty dimensions and density variations in the SiGe fin was demonstrated with our dedicated data treatment routine., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Using modern wide-angle atom probes, it is possible to acquire atomic scale 3D data containing 1000 s of nm(2) of interfaces. It is therefore possible to probe the distribution of segregated species across these interfaces. Here, we present techniques that allow the production of models for interfacial excess (IE) mapping and discuss the underlying considerations and sampling statistics. We also show, how the same principles can be used to achieve thickness mapping of thin films. We demonstrate the effectiveness on example applications, including the analysis of segregation to a phase boundary in stainless steel, segregation to a metal-ceramic interface and the assessment of thickness variations of the gate oxide in a fin-FET., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work we examine the role of lateral phase separation in cholesterol-containing biomimetic membranes on the disrupting action of melittin using a label-free surface-sensitive technique, quartz crystal microbalance with dissipation monitoring (QCM-D). Melittin disruption mechanisms depend strongly on the geometry of the lipid layer; however, despite the interplay between layer geometry/thickness and melittin activity, results indicate that the presence of lipid heterogeneity and lateral phase separation greatly influences the disrupting efficiency of melittin. In homogeneous non-raft forming membranes with high cholesterol content, melittin spontaneous activity is strongly delayed compared to heterogeneous raft-forming systems with the same amount of cholesterol. These results confirm the importance of lateral phase separation as a determinant factor in peptide activity. The information provided can be used for the design of more efficient antimicrobial peptides and the possibility of using a label-free approach for tailored-membranes and interactions with other types of peptides, such as amyloid peptides., (Copyright © 2014 Elsevier B.V. All rights reserved.)