Subject: Life Sciences, Biochemistry Keywords: super-resolution microscopy; advanced light microscopy; quantitative microscopy; live-cell microscopy; SMLM; STORM; SIM; STED; expansion microscopy; influenza virus; viral replication
Online: 6 January 2021 (10:40:59 CET)
With an estimated 3 to 5 million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society  and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are shedding new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies.
ARTICLE | doi:10.20944/preprints202210.0339.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: low-cost fluorescence microscopy; smartphone microscopy; 3D-printed devices; laser-based microscopy
Online: 21 October 2022 (13:48:31 CEST)
Fluorescence microscopy is an important tool for disease diagnosis, often requiring costly optical components, such as fluorescence filter cubes and high-power light sources. Due to its high cost, conventional fluorescence microscopy cannot be fully exploited in low-income settings. Smartphone-based fluorescence microscopy becomes an interesting low-cost alternative, but raises challenges in the optical system. We present the development of a low-cost inverted laser fluorescence microscope, that uses a smartphone to visualize the fluorescence image of biological samples. Our fluorescence microscope uses a laser-based simplified optical filter system, that provides analog optical filtering capabilities of a fluorescence filter cube. Firstly, we validated our inverted optical filtering by visualizing microbeads labeled with three different fluorescent compounds or fluorophores, commonly used for disease diagnosis. Secondly, we validated the disease diagnosis capabilities, by comparing the results of our device with those of a commercial fluorescence microscope. We successfully detected and visualized Trypanosoma cruzi parasites, responsible of the Chagas infectious disease, and the presence of Antineutrophil cytoplasmic antibodies of the ANCA non-communicable autoimmune disease. The samples were labeled with the fluorescein isothiocyanate (FITC) fluorophore, one of the most commonly used for disease diagnosis. Our device provides a 400 X magnification and is at least two orders magnitude cheaper than conventional commercial fluorescence microscopes.
REVIEW | doi:10.20944/preprints201803.0273.v1
Subject: Physical Sciences, Optics Keywords: Quantitative phase microscopy; Digital holographic microscopy; lensless microscopy; resolution enhancement; space-bandwidth product
Online: 30 March 2018 (14:09:57 CEST)
Quantitative phase microscopy (QPM), a technique combining phase imaging and microscopy, enables visualization of the 3-D topography in reflective samples as well as the inner structure or refractive index distribution of transparent and translucent samples. However, as in conventional optical microscopy, QPM provides either a large field of view (FOV) or a high resolution but not both. Many approaches such as oblique illumination, structured illumination and speckle illumination have been proposed to improve the spatial resolution of phase microscopy by restricting other degrees of freedom (mostly time). Therefore, the space bandwidth product (SBP) of QPM becomes enlarged. This paper aims to provide an up-to-date review on the resolution enhancement approaches of QPM, discussing the pros and cons of each technique as well as the confusion on resolution definition claim on QPM and other coherent microscopy.
ARTICLE | doi:10.20944/preprints202012.0302.v1
Subject: Materials Science, Biomaterials Keywords: X-ray Photoelectron Spectroscopy; Atomic Force Microscopy; Confocal Laser Scanning Microscopy; Scanning Electron Microscopy
Online: 13 December 2020 (21:34:33 CET)
Self-assembling films typically used for colloidal lithography have been applied to pine wood substrates to change the surface wettability. Therefore, monodisperse polystyrene (PS) spheres have been deposited onto a rough pine wood substrate via dip coating. The resulting PS sphere film resembled a polycrystalline FCC-like structure with typical domain sizes of 5 – 15 single spheres. This self-assembled coating was further functionalized via an O2 plasma. This plasma treatment strongly influenced the particle sizes in the outermost layer, and hydroxyl as well as carbonyl groups were introduced to the PS spheres’ surfaces, thus generating a superhydrophilic behaviour.
ARTICLE | doi:10.20944/preprints202207.0143.v1
Subject: Biology, Plant Sciences Keywords: pollination; pollen adhesion; pollenkitt; atomic force microscopy; cryogenic scanning electron microscopy; centrifugation
Online: 10 July 2022 (05:57:28 CEST)
Although pollination is one of the most crucial biological processes that ensures plant reproduction, its mechanisms are poorly understood. Especially in insect-mediated pollination, a pollen undergoes several attachment and detachment cycles when being transfered from anther to insect and from insect to stigma. The influence of the surface properties of pollen, insect and floral surfaces on the adhesion forces that mediate pollen transfer are poorly studied. Here, we investigate the adhesive properties of Hypochaeris radicata pollen and their dependence on pollen ageing by quantifying the pull-off forces from glass slides using centrifugation and atomic force microscopy. We found that the properties of the pollenkitt – the viscous, lipid liquid on the surface of most pollen grains – influences the forces necessary to detach a pollen from hydrophilic surfaces. Our results show that aged H. radicata pollen form weaker adhesions to hydrophilic glass than fresh ones. On the other hand, when a pollen grain ages in contact with glass, the adhesion between the two surfaces increases over time. This study shows for the first time the pollen ageing effect on the pollination mechanism.
ARTICLE | doi:10.20944/preprints201806.0309.v1
Subject: Materials Science, General Materials Science Keywords: metallic glasses; Ti-alloy; microstructure; scanning electron microscopy and transmission electron microscopy
Online: 20 June 2018 (03:31:13 CEST)
The present study investigation, our results on characterization of commercially pure-Ti alloy brazed with metallic glass ribbons of Ti20Zr20Cu60-xNix (x = 30, 40, and 50) metallic glass ribbons were produced using a vacuum melt spinner. These ribbons were then used as filler materials for vacuum brazing of two Ti alloy plates at 1268, 1277 and 1279 K for a period of 10 min. Field-Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM) and the energy dispersive X-ray spectroscopy (EDX). The as-spun ribbons showed fully amorphous structure when examined on both surfaces by XRD and also verified by TEM investigation. The brazing joint of two Ti-plates using the metallic glass ribbon when brazed with Ni50 was found to be of very high strength. FESEM characterization of the cross-section of the brazed joints shows sub-micron size grains uniformly distributed in the matrix with brighter appearance. FESEM and EDX analysis revealed that the sub-micron grains are rich in Ti & Ni while the matrix phase mainly consisted of Ti. BSE image along with EDS Analysis indicated that the brazed joint has a presence of NiTi2 and Cu2 (Ni Zr) phases which could be responsible for increase in the strength of the brazed joint.
ARTICLE | doi:10.20944/preprints202205.0405.v1
Subject: Engineering, Other Keywords: σ phase; high entropy alloy; X-Ray Diffraction; Scanning Electron Microscopy; Transmission Electron Microscopy
Online: 31 May 2022 (03:12:34 CEST)
In the perspective of designing complex concentrated alloys (CCAs) for structural applications, sigma (σ) phase is important. It is generally known to be an embrittling phase leading to a degradation in the mechanical properties of CCAs. This necessitates detailed investigations on the evolution of σ phase in these alloys. In the present study, an equiatomic CoCrCuMnNi CCA is chosen, in which σ phase appears at 800°C, as reported in a recent publication . To this end, the present investigation aims at understanding the evolution of this phase so that its presence can be controlled for the purpose of preventing degradation in mechanical properties. In the present work, a vacuum arc melted alloy, after homogenisation (at 400°C for 5h), is heat-treated for different times ranging from 1 to 9h at 800°C followed by water quenching. Extensive characterisation has been carried out using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques to understand the sequence of σ phase precipitation at 800°C.
ARTICLE | doi:10.20944/preprints202106.0639.v1
Subject: Life Sciences, Biochemistry Keywords: Microscopy; Bacterial cell biology; Nanofabrication; Microfluidics
Online: 28 June 2021 (10:38:11 CEST)
Light microscopy is indispensable for analysis of bacterial spatial organization. However, imaging in bacteria is difficult due to their small sizes and the fact that most species are non-spherical, meaning they typically lie horizontally on a microscope coverslip. This is especially problematic when considering that many essential bacterial processes—such as cell division—occur along the short axes of these cells, and so are viewed side-on by standard microscopy. We recently developed a pair of methods to overcome this problem by forcing cells to stand vertically during imaging, named VerCINI (Vertical Cell Imaging by Nanostructured Immobilisation) and µVerCINI (Microfluidic VerCINI). The concept behind both methods is that cells are imaged while confined vertically inside cell traps made from a nanofabricated mould. By doing so, the short axes of the cells are rotated parallel to the microscope imaging plane and are imaged with high resolution. μVerCINI combines the vertical cell confinement with microfluidics so that vertical imaging can be done during fluid exchange, such as during antibiotic perturbations. Here, we provide a practical guide to implementing both VerCINI and µVerCINI, with detailed protocols and experience-based tips so that interested researchers can easily use one or both imaging methods to complement their current approaches.
ARTICLE | doi:10.20944/preprints201908.0319.v1
Subject: Biology, Other Keywords: Phototoxicity, Photodamage, Super-Resolution Microscopy, Fluorescence
Online: 30 August 2019 (08:22:58 CEST)
Super-Resolution Microscopy enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we overview new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations.
Subject: Life Sciences, Cell & Developmental Biology Keywords: lamins; structured illumination microscopy; single molecule localization microscopy; steerable filters; computational geometry; delaunay triangulation; voronoi tessellation
Online: 22 March 2019 (15:55:59 CET)
The nuclear lamina consists of a dense fibrous meshwork made of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.
TECHNICAL NOTE | doi:10.20944/preprints202203.0146.v1
Subject: Life Sciences, Biophysics Keywords: expansion microscopy; yeast; Saccharomyces cerevisiae; super-resolution
Online: 10 March 2022 (10:51:02 CET)
The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross- linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.
ARTICLE | doi:10.20944/preprints201904.0169.v1
Subject: Medicine & Pharmacology, Dentistry Keywords: esthetic archwires; coating stability; backscattered electron microscopy
Online: 15 April 2019 (11:55:14 CEST)
Background/Aim: There is continuing interest in engineering esthetic labial archwires. The aim of this study was to coat nickel-titanium (NiTi) and beta-titanium (β-Ti), also known as titanium molybdenum (TMA), archwires by plasma electrolytic oxidation (PEO) and to analyze the characteristics of the PEO-surfaces. Materials and Methods: PEO-coatings were generated on 0.014-inch NiTi and 0.19x0.25-inch β-Ti archwires. The surfaces were analyzed by scanning electron microscopy and stereomicroscopy. Cytocombability testing was performed with ceramized and untreated samples according to EN ISO 10993-5 in XTT-, BrdU- and LDH-assays. The direct cell impact was analyzed using LIVE-/DEAD-staining. In addition, the archwires were inserted in an orthodontic model and photographs were taken before and after insertion. Results: The PEO coatings were 15 to 20 µm thick and esthetically pleasing. The cytocompatibility analysis revealed good cytocompatibility results for both ceramized NiTi and β-Ti archwires. In the direct cell tests, the ceramized samples showed improved compatibility as compared to those of uncoated samples. However, bending of the archwires resulted in loss of the PEO-surfaces. Nevertheless, it was possible to insert the β-Ti PEO-coated archwire in an orthodontic model without loss of the PEO-ceramic. Conclusion: PEO is a promising technique for the generation of esthetic orthodontic archwires. Since the PEO-coating does not resist bending, its clinical use seems to be limited so far to orthodontic techniques using straight or pre-bended archwires.
COMMUNICATION | doi:10.20944/preprints202104.0447.v1
Subject: Physical Sciences, Acoustics Keywords: optics; resolution; superresolution; Ernst Abbe; structured illumination; non-linear; image scanning microscopy; total internal reflection; MINFLUX; localization microscopy
Online: 16 April 2021 (14:45:48 CEST)
This article presents answers to the questions on superresolution and structured illumination microscopy as raised in the editorial of a recent publication [K. Prakash et al. arXiv, 2102.13649, 2021]. The answers are based on my personal views on superresolution in light microscopy, supported by reasoning. Discussed are the definition of superresolution, Abbe’s resolution limit and the classification of superresolution methods into non-linear-, prior-knowledge- and near-field-based superresolution. A further focus is put on capabilities and technical aspects of present and future structured illumination microscopy (SIM) methods.
TECHNICAL NOTE | doi:10.20944/preprints202111.0488.v2
Subject: Life Sciences, Microbiology Keywords: spheroid; tumour spheroid; mounting; clearing; microscopy; structure; confocal
Online: 31 March 2022 (09:57:33 CEST)
Tumour spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining high-quality data relating to the inner structure of spheroids is important for analysis, yet existing techniques often use equipment that is not commonly available, are expensive, laborious, cause significant size distortion, or are limited to relatively small spheroids. We present a high-throughput method of mounting, clearing, and imaging tumour spheroids that causes minimal size distortion. Spheroids are mounted in an agarose gel to prevent movement, cleared using a solution prepared from commonly available materials, and imaged using confocal microscopy. We find that our method yields high quality two- and three-dimensional images that provide information about the inner structure of spheroids.
REVIEW | doi:10.20944/preprints202202.0096.v1
Subject: Materials Science, Metallurgy Keywords: Grain boundaries; solute decoration; correlative microscopy; cohesive strength
Online: 7 February 2022 (16:05:39 CET)
Solute decoration at grain boundaries (GB) leads to a number of phenomenon such as changes in interface structure, mobility, cohesion etc. Recent experimental investigations on interfacial segregation in steels are based on microstructural characterisation using two correlative methodologies, namely, Transmission Electron Microscopy-Atom Probe Tomography (APT) and Electron Backscatter Diffraction-APT. Considering the growing interest in this avenue, the present review is aimed at addressing the common adsorption isotherms used for quantifying interfacial segregation and providing an overview of the present state of experimental research in the area of GB segregation in steels. The areas where an understanding of GB segregation may be utilised have also been highlighted with a focus on the experimental challenges associated with understanding GB segregation in steels.
ARTICLE | doi:10.20944/preprints202106.0699.v1
Subject: Biology, Anatomy & Morphology Keywords: pollen, pollen classes, determination, 3D, laser scanning microscopy
Online: 29 June 2021 (11:45:54 CEST)
Pollen analysis as a part of palynology deals with the morphological determination of pollen and spores. Different technologies with different resolutions varying from simple light microscopy to highly elaborate electron microscopy are used for the examination, depending on the area of application (e.g. sedimentology, melissopalynology, forensic palynology, etc.). To answer the question of whether laser scanning microscopy (LSM) can replace scanning electron microscopy (SEM) for the determination of pollen species, 168 species were examined using LSM. It was concluded that LSM is both efficient and easy to handle. After preparing the fresh pollen, a 3D laser scan takes 5-10 minutes and unlike using SEM, the pollen does not have to be sputtered or processed. The 3D scans can be measured quickly and easily with the integrated software and there were no observable artifacts. At magnifications up to 8545x, the image quality is comparable to that of a sputtered SEM sample whereas at higher magnifications, the SEM method is superior. Overall, pollen display by LSM is much less time consuming and more cost effective than with the SEM method.
REVIEW | doi:10.20944/preprints202105.0352.v1
Subject: Life Sciences, Biochemistry Keywords: 3d printing; microscopy; open-source; optics; super-resolution
Online: 14 May 2021 (16:10:24 CEST)
The maker movement has reached the optics labs, empowering researchers to actively create and modify microscope designs and imaging accessories. 3D printing has especially had a disruptive impact on the field, as it entails an accessible new approach in fabrication technologies, namely additive manufacturing, making prototyping in the lab available at low cost. Examples of this trend are taking advantage of the easy availability of 3D printing technology. For example, inexpensive microscopes for education have been designed, such as the FlyPi. Also, the highly complex robotic microscope OpenFlexure represents a clear desire for the democratisation of this technology. 3D printing facilitates new and powerful approaches to science and promotes collaboration between researchers, as 3D designs are easily shared. This holds the unique possibility to extend the open-access concept from knowledge to technology, allowing researchers from everywhere to use and extend model structures. Here we present a review of additive manufacturing applications in microscopy, guiding the user through this new and exciting technology and providing a starting point to anyone willing to employ this versatile and powerful new tool.
Subject: Medicine & Pharmacology, Allergology Keywords: fire-related death; viability; light microscopy; bronchiolar epithelium
Online: 25 February 2021 (13:12:51 CET)
Fire-related deaths are issues for forensic pathologists particularly in ascertaining if death occurred before or during fire. The authors highlight a unique bronchiolar epithelial cytological clue (nuclear heat-induced elongation) determined by active inhalation of hot gases and fumes, not yet described in the literature at distal small airways level.
COMMUNICATION | doi:10.20944/preprints202011.0042.v1
Subject: Biology, Ecology Keywords: compound microscope; inexpensive microscopy; ultra-low-cost microscope
Online: 2 November 2020 (13:36:42 CET)
We report a very frugal microscope building method from easily realizable parts. We show a highly portable field ready compound type microscope with bright field, dark field and projection microscopy capabilities. It has good resolution and magnification for multiple aspects of education and diagnosis. We demonstrate that such a system can be built from simple lenses in laser pointers and camera modules with no specific 3d printed parts or costly lenses. We show all the parts of the system like stage, coarse and fine focussing system, microscope body, even slides can be built from commonly available materials like soda bottle and DVD disks. We list alternate, safer and easily available staining methods and chemicals. The microscope is developed with a target that it can be self-developed even in rural areas of the world with only resources at one’s disposal with very little education. This features a stable focussing and panning system for comfortable viewing through eyes and also easy imaging with a smartphone. We show its utility for microorganism analysis and potential for clinical diagnosis.
ARTICLE | doi:10.20944/preprints202004.0296.v1
Online: 17 April 2020 (08:30:13 CEST)
Collective spread of aggregated viral particles may have beneficial effects on viral capability to survive in the external environment, to counteract immune responses, and to successfully colonize host cells. Here we ask whether SARS-Cov-2 particles, responsible for COVID-19, display collective clustering behavior. Looking at microphotographs and movies of SARS-Cov-2 particles emerging from the surface of cultured cells, we describe single virions that tend to aggregate in progressively larger globular assemblies, until a network-like appearance is achieved. When SARS-Cov-2 particles stick into each other, the squeezing of single virions leads to improved viral package in host’s fluids. We discuss how these findings might explain both the ability to spread of SARS-Cov-2 and the clinical severity of COVID-19 in humans, paving the way to novel therapeutic strategies to mechanically disrupt collective clustering.
REVIEW | doi:10.20944/preprints201811.0596.v1
Subject: Biology, Other Keywords: HIV-1 assembly, Gag, single molecule microscopy, dynamics
Online: 26 November 2018 (14:12:08 CET)
HIV-1 assembly is a complex mechanism taking place at the plasma membrane of the host cell. It requires nice spatial and temporal coordination to end up with a full immature virus. Researchers have extensively studied HIV-1 assembly molecular mechanism during the past decades, in order to dissect the respective roles of viral proteins, viral genome and host cell factors. Nevertheless, the time course of the process has been observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution now permit to study assemblies and their consequences at the single molecule level within (living) cells. In this review, after a short description of these new approaches, we will show how HIV-1 assembly in cells has been revisited using these advanced super resolution microscopy techniques and how much it could make a bridge in studying assembly from the single molecule to the host cell.
ARTICLE | doi:10.20944/preprints201810.0248.v2
Subject: Materials Science, Surfaces, Coatings & Films Keywords: low temperature carburizing; XRD; optical microscopy; carburized layer
Online: 22 October 2018 (11:19:18 CEST)
Carburizing of stainless steels at low temperatures (below 500°C) develops a high content carbon layer, known for its high hardness. X-ray Diffraction investigation indicates that carburization treatment does not impact the structure of substrate; however, introduced carbon causes expansion in the carburized layer through the increase in d-spacing. Characterization of carbon concentration and hardness profiles indicate that carbon content gradually decreases while moving further into the substrate; and the origin of the increased hardness of the expanded layer arises from the super-saturated carbon content, following the solid solution strengthening theory. An area that is not well understood is in regard to the carburized layer’s relation to the substrate and the significance in their differences. Using two grades of stainless steel, AISI 316L and PH 17-4, it was observed that the carburized layer is not a separate layer, but a higher carbon content version of the substrate,
ARTICLE | doi:10.20944/preprints201806.0303.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: photothermal microscopy; label-free imaging; melanoma; texture analysis
Online: 19 June 2018 (15:28:53 CEST)
Label-free confocal photothermal (CPT) microscopy was utilized for the first time to investigate malignancy in mouse skin cells. A laser diode (LD) with 405nm or 488nm was used as a pump and 638nm LD as a probe for the CPT microscope. The Grey Level Cooccurrence Matrix (GLCM) for texture analysis was applied to the CPT images. Nine parameters of GLCM were calculated for the intracellular super-resolved CPT images, and the parameters Entropy and Prominence were found to be most suited among the nine parameters to discriminate between healthy cells and MM cells in case pump wavelength of 488nm is used.
BRIEF REPORT | doi:10.20944/preprints201806.0086.v1
Subject: Medicine & Pharmacology, Dentistry Keywords: er:yag laser; debonding; laminate veneer; scanning electron microscopy
Online: 6 June 2018 (11:53:01 CEST)
The purpose of this study consists to verify if Er:YAG laser, at low fluences, is able to debond porcelain veneers, successfully used to improve anterior tooth esthetics, without damaging the tooth structures. A total of twelve freshly extracted teeth were used and samples were decontaminated, stored, prepared to obtain Veneers adhesion. One week after, Er:YAG laser with a non-contact sapphire tip with air-water spray was used for veneer debonding at 100 mJ of power and 30Hz of frequency (Fluence 5.91 J/cm2). Results demonstrated that veneer debonding is possible with an Er:YAG laser and that the total number of pulses does not seem related to its efficiency. SEM observation confirms that residual tooth structure is not altered when using these low fluences. Low fluences with Er:YAG laser are able to debond veneers while preserving the tooth structures. SEM observation confirmed that residual tooth structure is not altered with low fluences.
ARTICLE | doi:10.20944/preprints202211.0199.v1
Subject: Biology, Plant Sciences Keywords: cyanobacteria; photosystem; fast fluorescence kinetics; optical microcavity; fluorescence microscopy
Online: 10 November 2022 (11:05:24 CET)
Photosynthesis is one the most important biological processes on earth, producing life-giving oxygen and is the basis for a large variety of plant products. Measurable properties of photosynthesis provide information about its biophysical state and, in turn, the physiological conditions of a photoautotrophic organism. For instance, chlorophyll fluorescence of an intact photosystem is not linear as in the case of a single fluorescent dye in solution, but shows temporal changes related to the quantum yield of the photosystem. Commercial photosystem analyzers already use the fluorescence kinetics characteristics of photosystems to infer the viability of organisms under investigation. Here, we provide a novel approach based on an optical Fabry-Pérot microcavity or that enables the readout of photosynthetic properties and activity for an individual cyanobacterium. This approach offers a completely new dimension of information, which would normally be lost due to averaging in ensemble measurements obtained from a large population of bacteria.
ARTICLE | doi:10.20944/preprints202209.0203.v1
Subject: Biology, Ecology Keywords: slide scanning; Bacillariophyceae; method comparison; image annotation; light microscopy
Online: 14 September 2022 (09:16:07 CEST)
Diatom identification and counting by light microscopy is a fundamental method in ecological and water quality investigations. Here we present a new variant of this method based on “digital virtual slides”, and compare it to the traditional, non-digitized light microscopy workflow. We analysed three replicates of six samples using two methods: 1) working directly on a light microscope (the “traditional” counting method), and 2) preparing “virtual digital slides” by high-resolution slide scanning and subsequently identifying and labelling individual valves or frustules using a web browser-based image annotation platform (the digital method). Both methods led to comparable results in terms of species richness, diatom indices and diatom community composition. Although counting by digital microscopy was slightly more time consuming, our experience points out that the digital workflow can not only improve the transparency and reusability of diatom counts but it can also increase taxonomic precision. The introduced digital workflow can also be applied for taxonomic inter-expert calibration through the web, and for producing training image sets for deep-learning-based diatom identification, making it a promising and versatile alternative or extension to traditional light microscopic diatom analyses in the future.
ARTICLE | doi:10.20944/preprints202204.0209.v1
Subject: Physical Sciences, Condensed Matter Physics Keywords: defect-induced superconductivity; graphite; stacking faults; magnetic force microscopy
Online: 22 April 2022 (03:43:45 CEST)
Granular superconductivity at high temperatures in graphite can emerge at certain two-dimensional (2D) stacking faults (SFs) between regions with twisted (around the c-axis) or untwisted crystalline regions with Bernal (ABA...) and/or rhombohedral (ABCABCA...) stacking order. One way to observe experimentally such 2D superconductivity is to measure the frozen magnetic flux produced by a permanent current loop that remains after removing an external magnetic field applied normal to the SFs. Magnetic force microscopy was used to localize and characterize such a permanent current path found in one natural graphite sample out of ∼50 measured graphite samples of different origins. The position of the current path drifts with time and roughly follows a logarithmic time dependence similar to the one for flux creep in type II superconductors. We demonstrate that a ≃10nm deep scratch on the sample surface at the position of the current path causes a change in its location. A further scratch was enough to irreversibly destroy the remanent state of the sample at room temperature. Our studies clarify some of the reasons for the difficulties of finding a trapped flux in remanent state at room temperature in graphite samples with SFs.
Subject: Physical Sciences, Optics Keywords: Interference microscopy; coherence scanning interferometry; three-dimensional transfer function
Online: 15 September 2021 (12:46:05 CEST)
Three-dimensional transfer functions (3D TFs) are generally assumed to fully describe the transfer behavior of optical topography measuring instruments such as coherence scanning interferometers in the spatial frequency domain. Therefore, 3D TFs are supposed to be independent of the surface under investigation resulting in a clear separation of surface properties and transfer characteristics. In this paper we show that the 3D TF of an interference microscope differs depending on whether the object is specularly reflecting or consists of point scatterers. In addition to the 3D TF of a point scatterer, we will derive an analytical expression for the 3D TF corresponding to specular surfaces and demonstrate this as being most relevant in practical applications of coherence scanning interferometry (CSI). We additionally study the effects of temporal coherence and disclose, that in conventional CSI temporal coherence effects dominate. However, narrowband light sources are advantageous if high spatial frequency components of weak phase objects are to be resolved, whereas for low-frequency phase objects of higher amplitude the temporal coherence is less affecting. Finally, we present an approach that explains the different transfer characteristics of coherence peak and phase detection in CSI signal analysis.
ARTICLE | doi:10.20944/preprints202104.0573.v1
Subject: Engineering, Automotive Engineering Keywords: fluorescence microscopy; fluorescence emission, malignant tumor, diagnosis, animal experiment
Online: 21 April 2021 (11:47:14 CEST)
A surgical microscope is large in size, which makes it impossible to be portable. The distance between the surgical microscope and the observation tissue is 15–30 cm, and the adjustment range of the right and left of the camera is a maximum of 30°. Therefore, the surgical microscope is generated attenuation (above 58%) of irradiation optical source owing to the long working distance. Moreover, the observation of tissue is affected because of dazzling by ambient light as the optical source power is strong (55 to 160 mW/cm2). Further, observation blind spot phenomena will occur due to the limitations in adjusting the right and left of the camera. Therefore, it is difficult to clearly observe the tumor. In this study, a compact pen-type probe with a portable surgical microscope is presented. The proposed surgical microscope comprises a small and portable pen-type probe that can adjust the working distance between the probe and the observed tissue. In addition, it allows the adjustment of the viewing angle and fluorescence brightness. The proposed probe has no blind spots or optical density loss.
ARTICLE | doi:10.20944/preprints202104.0557.v1
Subject: Engineering, Automotive Engineering Keywords: fluorescence microscopy; fluorescence emission; malignant tumor; diagnosis; animal experiment
Online: 21 April 2021 (08:30:11 CEST)
A surgical microscope is large in size, making portability impossible. The distance between the surgical microscope and the observation tissue is 15 to 30 cm, while the maximum adjustment range of the camera to the right and left is 30°. Therefore, surgical microscopes cause attenuation (above 58%) of the irradiation optical source owing to the long working distance. Moreover, the observation of tissue was dazzled with ambient light because the optical power source was strong (50 to 160 mW/cm2). Owing to the limited ability to adjust the camera to the right and left, a blind spot occurs with a surgical microscope. Therefore, it is difficult to clearly observe a tumor. In this study, a compact pen-type probe with a portable surgical microscope is proposed. The pen-type probe is small with a portable shape, and is capable of adjusting the working distance between itself and the observed tissue. It is also possible to adjust the viewing angle and fluorescence brightness. The proposed pen-type probe has no blind spots or optical density loss.
ARTICLE | doi:10.20944/preprints202101.0635.v1
Online: 29 January 2021 (17:21:56 CET)
This paper presents an advanced microstructural analysis of the AlSiMg, Ti64 and N700 powders used for additive manufacturing. The internal microstructure of the regular and irregular powder grains were characterized down to atomic resolution by using scanning electron microscopy and high resolution scanning transmission electron microscopy.The accretionary forms on top of the irregular AlSiMg powder grains exhibit a slightly coarse microstructure with a network of eutectic Si consisting of nano-crystallites, suggestinga slower cooling than the grain itself that contain a predominately amorphous Si network. A nm thin amorphous C layer on the surface of some Ti64 plasma atomized powder grains promoted the attachment of satellites and growth of envelopes. In case of gas atomized N700 powder grains, we identified thin oxide and carbon amorphous layers as well as metal segregations at the interface between the grain body and the accretionary forms.
ARTICLE | doi:10.20944/preprints202006.0094.v1
Subject: Medicine & Pharmacology, General Medical Research Keywords: CCCP; Mitophagy; Regulatory T cells; Flow cytometry; fluorescence microscopy
Online: 7 June 2020 (15:03:23 CEST)
Objective: To investigate the effects and mechanisms of different concentrations of CCCP on mitophagy in human peripheral blood regulatory T cells. Methods: Tregs were isolated, identified and then grouped, treating with CCCP at a concentration of 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM for 24h in an incubator. Flow cytometry detected the reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial quality, and fluorescence microscopy observed the co-localization of mitochondria and lysosomes in each group. Results: The purity of CD4+CD25+Tregs was (93.36 ± 1.87) %. With the increase of CCCP concentration, the level of ROS gradually increased, while the MMP decreased gradually. About the mitochondria and lysosome fusion, the fluorescence intensity of orange (yellow) was the highest when the concentration of CCCP was in the range of 5-10 μM while decreased with the CCCP concentration continually increasing. The mitochondrial quality decreased with the increase of CCCP concentration. However, there was no significant difference between groups C, D and E. The mitochondrial quality of groups F and G were significantly lower than that of group E. Conclusions: With the concentration of CCCP gradually increased, the level of ROS in Treg cells increased, and MMP decreased, which promoted the mitophagy, mitochondrial quality maintains homeostasis; When ROS accumulated, and MMP decreased significantly, the mitophagy was inhibited, and the mitochondrial quality was significantly decreased.
ARTICLE | doi:10.20944/preprints202005.0119.v1
Subject: Life Sciences, Biophysics Keywords: pore-forming toxins; calcium; high-speed atomic force microscopy
Online: 7 May 2020 (10:23:54 CEST)
Pore Forming Toxins (PFTs), formed mainly by virulence factors of bacteria, belongs to Pore Forming Protein (PFP) family. Secreted as soluble monomers, they bind specific targets in membranes where their oligomerization and insertion place. Lysenin, a member of the PFTs, forms and oligomer after sphingomyelin binding, the so-called prepore, which become inserted forming a pore after a conformational change triggered by a pH decrease. In crowded conditions, oligomers tends to stay in prepore form because the prepore-to-pore transition is sterically blocked. In this study, we investigate the effect of calcium ions in those crowded conditions, finding that calcium act as a trigger for lysenin insertion. We localize the residues responsible for calcium sensitivity in a small α-helix. Our results are not only one of the few complete structural descriptions of prepore-to-pore transitions but the very first that involves a calcium triggering mechanism. The presence of glutamic or aspartic acids in the insertion domains could be an indication that calcium may be a general trigger for PFTs and more generally PFP.
ARTICLE | doi:10.20944/preprints201910.0210.v1
Subject: Physical Sciences, Applied Physics Keywords: scanning magnetic microscopy; Hall sensor; magnetic measurements; geological sample
Online: 18 October 2019 (08:53:15 CEST)
Scanning magnetic microscopy is a new tool that has recently been used to map magnetic fields with good spatial resolution and field sensitivity. This technology has great advantages over other instruments; for example, its operation does not require cryogenic technology, which reduces its operational cost and complexity. Here, we describe the construction of a customizing scanning magnetic microscope based on commercial Hall-effect sensors at room temperature that achieves a spatial resolution of 200 µm. Two scanning stages on the x- and y-axes of precision, consisting of two coupled actuators, control the position of the sample, and this microscope can operate inside or outside a magnetic shield. We obtained magnetic field sensitivities better than 521 nTrms/√Hz between 1 and 10 Hz, which correspond to a magnetic momentum sensitivity of 9.20 × 10–10 Am2. In order to demonstrate the capability of the microscopy, polished thin sections of geological samples, samples containing microparticles and magnetic nanoparticles were measured. For the geological samples, a theoretical model was adapted from the magnetic maps obtained by the equipment. Vector field maps are valuable tools for the magnetic interpretation of samples with a high spatial variability of magnetization. These maps can provide comprehensive information regarding the spatial distribution of magnetic carriers. In addition, this model may be useful for characterizing isolated areas over samples or investigating the spatial magnetization distribution of bulk samples at the micro and millimeter scales. As an auxiliary technique, a magnetic sweep map was created using Raman spectroscopy; this map allowed the verification of different minerals in the samples. This equipment can be useful for many applications that require samples that need to be mapped without a magnetic field at room temperature, including rock magnetism, the nondestructive testing of steel materials and the testing of biological samples. The equipment can not only be used in cutting-edge research but also serve as a teaching tool to introduce undergraduate, master's and Ph.D. students to the measurement methods and processing techniques used in scanning magnetic microscopy.
REVIEW | doi:10.20944/preprints201811.0543.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: hydroxyurea; cerebellum; neuron; immunohistochemistry; electron microscopy; cell death; apoptosis
Online: 22 November 2018 (07:09:28 CET)
The cytostatic agent hydroxyurea (HU) has proven to be beneficial for a variety of conditions in the disciplines of oncology, hematology, infectious disease and dermatology. It disrupts the S-phase of the cell cycle by inhibiting the ribonucleotide reductase enzyme, thus blocking the transformation of ribonucleotides into deoxyribonucleotides, a rate limiting step in DNA synthesis. HU is listed as an essential medicine by the World Health Organization. Several studies have indicated that HU is well tolerated and safe in pregnant women and very young pediatric patients. To our knowledge, only a few controlled studies about the adverse effects of HU therapy have been done in humans. Despite this, the prevalence of central nervous system abnormalities, including ischemic lesions and stenosis have been reported. This review will summarize and present the effects of HU-exposure on the prenatal and perinatal development of the rat cerebellar cortex and deep cerebellar nuclei neurons. Our results call for the necessity to better understand HU effects and define the administration of this drug to gestating women and young pediatric patients.
ARTICLE | doi:10.20944/preprints201808.0286.v1
Subject: Materials Science, Metallurgy Keywords: Thread fracture; tool wear; Taylor Equation; Scanning Electron Microscopy
Online: 16 August 2018 (13:59:10 CEST)
The diameter of a H13 steel tool with M6 threads and a pin diameter of 5.9 mm and a pin length of 5 mm was measured after each 25.4 mm length of friction stir processing (FSP) of 6061-T6 extrusions. The change in pin diameter with FSP time or distance did not exhibit any steady state and was found to have two distinct regions. Metallographic analysis of two tools subjected to FSP for 60 and 120 seconds showed that (i) threads fractured in early stages of FSP, (ii) a built-up layer formed between the threads, and (iii) threads progressively wore with processing time. The metallographic analysis of an embedded tool showed the presence of a fractured piece of the tool in the stir zone. These points are discussed in detail in the paper.
REVIEW | doi:10.20944/preprints201805.0142.v2
Subject: Materials Science, Nanotechnology Keywords: graphene; cold field emission; single-tip cathode; electron microscopy
Online: 28 May 2018 (09:02:44 CEST)
Although good field emission from graphene has been demonstrated from a wide variety of different microfabricated structures, very few of them can be used to improve the design of cold field emitters for electron microscopy applications. Most of them consist of densely packed nano-emitters, which produce a large array of defocused overlapping electron beams, and therefore cannot be subsequently focused down to a single nanometer electron probe. This paper reviews the kind of single-tip cathode structures suitable in cold field emission guns for instruments such as scanning electron microscopy, transmission electron microscope or scanning transmission electron microscopy, and reviews progress in fabricating them from graphene-based materials.
ARTICLE | doi:10.20944/preprints201805.0051.v1
Subject: Engineering, Mechanical Engineering Keywords: porous media; optical video microscopy; microfluidics; waterflooding; surfactants; polymers
Online: 3 May 2018 (05:52:37 CEST)
In this study, we examine microscale waterflooding in a randomly close-packed porous medium. Three different porosities are prepared in a microfluidic platform and saturated with silicone oil. Optical video fluorescence microscopy is used to track the water front as it flows through the porous packed bed. The degree of water saturation is compared to water containing two different types of chemical modifiers, sodium dodecyl sulfate (SDS) and polyvinylpyrrolidone (PVP), with water in the absence of a surfactant used as a control. Image analysis of our video data yield saturation curves and calculate fractal dimension, which we use to identify how morphology changes the way an invading water phase moves through the porous media. An inverse analysis based on the implicit pressure explicit saturation (IMPES) simulation technique uses mobility ratio as an adjustable parameter to fit our experimental saturation curves. The results from our inverse analysis combined with our image analysis show that this platform can be used to evaluate the effectiveness of surfactants or polymers as additives for enhancing the transport of water through an oil-saturated porous medium.
ARTICLE | doi:10.20944/preprints201708.0028.v1
Subject: Physical Sciences, Condensed Matter Physics Keywords: crystal growth; calcite; microfluidic; nanoconfinement; reflection interference contrast microscopy.
Online: 8 August 2017 (08:09:11 CEST)
Slow growth of calcite in confinement is abundant in Nature and man made materials. There is ample evidence that such confined growth may create forces that fracture solids. The thermodynamic limits are well known but since confined crystal growth is transport limited and difficult to control in experiment we have almost no information on the mechanisms or limits of these processes. We present a novel approach to in situ study of confined crystal growth using microfluidics for accurate control of the saturation state of the fluid and interferometric measurement of the topography of the growing confined crystal surface. We observe and explain the diffusion limited confined growth structures observed and can measure the crystal "floating" on a fluid film of 10-40~nm thickness due to the disjoining pressure. We find that there are two end member behaviours: smooth or intermittent growth in the contact region, the latter being faster than the former.
ARTICLE | doi:10.20944/preprints201706.0071.v1
Subject: Life Sciences, Biochemistry Keywords: 8-prenylnaringenin; naringenin; cellular accumulation; glioblastoma; cytotoxicity; confocal microscopy
Online: 16 June 2017 (03:25:34 CEST)
Gliomas are one of the most aggressive and treatment-resistant types of human cancer. One of the most promising field in gliomas cancer therapy is identification and evaluation of anticancer properties of compounds found in plants i.a. naringenin (N) and 8-prenylnaringenin (8PN). The prenyl group seem to be crucial to the anticancer activity of flavones, which may lead to enhanced cell membrane targeting and thus increased intracellular activity. Unfortunately, 8PN content in hop cones is from 10 to 100 times lower compared to other flavonoids i.e. xanthohumol. In this study we used a simple method for the synthesis of 8PN from isoxanthohumol, via O-demethylation with high, 97% of the isolated yield. Cellular accumulation and cytotoxicity of naringenin and 8-prenylnaringenin in normal (BJ) and cancer cells (U-118 MG) were also examined. Obtained data indicated that 8-prenylnaringenin exhibited higher toxicity against used cell lines than naringenin and both flavones inhibited stronger glioblastoma U-118 MG cells than normal fibroblasts. The anticancer properties of 8PN correlated with its significantly greater (37%), accumulation in glioblastoma cells than in normal fibroblasts. Additionally, naringenin indicated higher selectivity for glioblastoma as it was over 6 times more toxic for cancer than normal cells. Our results provide evidence that examined prenylated and non-prenylated flavanones have different biological activity against normal and cancer cell lines and this phenomenon may be useful in clinical practice to construct new, anticancer drugs for glioblastoma.
COMMUNICATION | doi:10.20944/preprints201610.0058.v2
Subject: Materials Science, Surfaces, Coatings & Films Keywords: CuInSe2 electrodeposition; alkaline doping; current sensing atomic force microscopy
Online: 18 December 2016 (09:48:30 CET)
The local electrical response in alkaline-doped CuInSe2 films prepared by single step electrodeposition onto Cu substrates was studied by current sensing atomic force microscopy. The CIS films were prepared from single baths containing the dopant ions (Li, Na, K or Cs) and were studied by X-ray diffraction, scanning electron microscopy, energy dispersive X-ray spectroscopy and photocurrent response. Increased crystallinity and surface texturing as the ion size increases was observed as well as enhanced photocurrent response in Cs doped CIS. Li and Na doped films have larger conductivity than the undoped film while the K and Cs doped samples display shorter currents and the current images indicate strong charge accumulation in the K and Cs doped films forming surface capacitors. Corrected CAFM IV curves were adjusted with Shockley equation.
COMMUNICATION | doi:10.20944/preprints202111.0148.v1
Subject: Materials Science, Biomaterials Keywords: Biocompatibility; Biodegradation; Biomaterial; Polylactide-based materials; Polymer; Scanning electron microscopy
Online: 8 November 2021 (13:43:50 CET)
In horses, there is an increasing interest in developing long-lasting drug formulations, with bi-opolymers as viable carrier alternatives in addition to their use as scaffolds, suture threads, screws, pins, and plates for orthopedic surgeries. This communication focuses on the prolonged biocompatibility and biodegradation of PLA, prepared by hot pressing at 180 ºC. Six samples were implanted subcutaneously on the lateral surface of the neck of one horse. The polymers remained implanted for 24 to 57 weeks. Physical examination, plasma fibrinogen, and the mechanical noci-ceptive threshold (MNT) were performed. After 24, 28, 34, 38, and 57 weeks, the materials were removed for histochemical analysis using hematoxylin-eosin and scanning electron microscopy (SEM). There were no essential clinical changes. MNT decreased after the implantation procedure, returning to normal after 48h. A foreign body response was observed by histopathologic evalua-tion up to 38 weeks. At 57 weeks, no polymer or fibrotic capsules were identified. SEM showed surface roughness suggesting a biodegradation process, with an increase in the average pore di-ameter. As in the histopathological evaluation, it was not possible to detect the polymer 57 weeks after implantation. PLA showed biocompatible degradation and these findings may contribute to future research in the biomedical area.
ARTICLE | doi:10.20944/preprints202106.0052.v1
Subject: Life Sciences, Biochemistry Keywords: antibiotics; conventional; microbiology; microfluidics; microscopy; mycobacterium smegmatis; population; single cell
Online: 2 June 2021 (08:32:03 CEST)
To reveal rare phenotypes in bacterial populations conventional microbiology tools should be advanced to generate rapid, quantitative, accurate and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells we applied both conventional assays and a microfluidic-microscopy method to reveal antibiotic-tolerance mechanisms of wild type and the msm2570::Tnmutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared with wild-type cells. This is the first study that characterized the antibiotic-tolerance phenotype of the msm2570::Tn mutant that has a transposon insertion in the msm2570 gene encoding a putative xanthine/uracil permease, which enrolls in uptake of nitrogen compound during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particularly, antibiotics those targets cell wall integrity.
Subject: Keywords: Cellulose Nanocrystal; Anthraquinone; Reactive Oxygen Species; FTIR; Atomic Force Microscopy
Online: 29 July 2020 (10:59:57 CEST)
Antimicrobial and anti-viral materials have attracted significant interest in recent years due to increasing occurrences of nosocomial infections and pathogenic microbial contamination. One method to address this is the combination of photoactive compounds that can produce reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals to disinfect microbes, with carrier materials that meet the application requirements. Using anthraquinone (AQ) and cellulose nanocrystals (CNCs) as the photoactive and carrier components respectively, this work demonstrates the first covalent incorporation of AQ onto CNCs. The morphology and the photoactive properties were investigated, revealing the structural integrity of the CNCs, and high degree of photoactivity of AQ-CNC materials upon UVA exposure. The AQ-CNCs also exhibited an unexpected persistent generation of ROS under darkness, which adds advantages for antimicrobial applications.
ARTICLE | doi:10.20944/preprints202003.0224.v1
Subject: Medicine & Pharmacology, Dentistry Keywords: fibrin matrix; g-force; growth factors; scanning electron microscopy; fibrinolysis
Online: 13 March 2020 (03:05:18 CET)
Blood Concentrates (BCs) are autologous non-transfusional therapeutical preparations with biological properties applied in tissue regeneration. These BCs differ in the preparation method, in fibrin network architecture, growth factors release as well as in platelet/cell content. Methodological changes result in distinct matrices that can compromise their clinical effectiveness. The present study evaluated the influence of different g-forces and types of tubes in the release of vascular endothelial growth factor (VEGF) from platelet-rich fibrin (PRF) as a function of time. The PRF-like samples were obtained with three g-forces (200, 400, and 800 x g) for 10 minutes in pure glass tubes or in polystyrene-clot activator tubes. Scanning and Transmission electron microscopy was used to morphometric analyzes of PRF’s specimens and flow cytometry was used to quantify VEGF slow release until 7 days. Our results showed that platelets were intact and adhered to the fibrin network, emitting pseudopods and in degranulation. The fibrin network was rough and twisted with exosomic granulations impregnated on its surface. An increase in the concentration of VEGF in the PRF supernatant was observed until 7 days for all g forces (200, 400 or 800 x g), with the highest concentrations observed with 200 x g, in both tubes, glass or plastic. Morphological analyzes showed a reduction in the diameter of the PRF fibers after 7 days. Our results showed that g-force interferes with the shape of the fibrin network in the PRF, as well as affect the release of VEGF stored into platelets. This finding may be useful in applying PRF to skin lesions, in which the rapid release of growth factors can favor the tissue repair process. Our observations point to a greater clarification on the methodological variations related to obtaining PRF matrices, as they can generate products with different characteristics and degrees of effectiveness in specific applications.
ARTICLE | doi:10.20944/preprints201911.0124.v1
Subject: Engineering, Industrial & Manufacturing Engineering Keywords: additive manufacturing; confocal microscopy; measurement; calibration; traceability; uncertainty; quality assessment
Online: 12 November 2019 (07:56:55 CET)
Additive manufacturing (AM) is a promising new technology that is having a very fast growth from home workshops to high-tech cutting-edge factories. As any manufacturing technique, adequate metrology services are needed to assure the quality of items manufactured by AM. One of the most widely used instruments to measure the characteristics of surfaces manufactured with AM is the confocal microscope. In this paper, authors present a whole calibration procedure for confocal microscopes designed to be implemented preferably in workshops or industrial environments rather than in research and development departments. Because of that, it is as simple as possible. The procedure is designed without forgetting any of the key aspects that need to be taken into account and based on classical reference material standards. These standards can be easily found in industrial dimensional laboratories and easily calibrated in accredited calibration laboratories.
ARTICLE | doi:10.20944/preprints201910.0117.v1
Subject: Engineering, Industrial & Manufacturing Engineering Keywords: SART process; precipitation aggregates; image analysis; microscopy; particle size distribution
Online: 10 October 2019 (10:55:10 CEST)
Precipitation processes are technologies commonly used in hydrometallurgical plants to recover metals or to treat wastewaters. Moreover, solid-liquid separation technologies, such as thickening or filtering, are relevant unit operations, included in the precipitation technologies. These methods are strongly dependent on the characteristics of the solid precipitates formed during the specific precipitation reaction. One of these characteristics is the particle size distribution (PSD) of the solid precipitates which are fed into a solid-liquid separation process. Therefore, PSD determination is a typical practice for the characterization of the slurries generated in a precipitation plant. Furthermore, the precipitates generated in these processes have a colloidal or aggregation behavior, depending on the operational conditions. Nevertheless, the conventional methods used to estimate PSD (e.g., laser diffraction and/or ciclosizer) have not been designed to measure particles that tend to aggregate or disaggregate, since they include external forces (e.g., centrifugal, agitation, pumping and sonication). These forces affect the true size of the aggregates formed in a unit operation, thereby losing representativity in terms of aggregates particle size. This study presents an alternative method of measuring the size distribution of particles with aggregation behavior, particularly, by using non-invasive microscopy and image processing and analysis. The samples used have been obtained from an experimental precipitation process by applying sulfidization to treat the cyanide-copper complexes contained in a cyanidation solution. This method has been validated with statistical tools and compared with a conventional analysis based on laser diffraction. Our results show significant differences between the methods analyzed, demonstrating that image processing and analysis by microscopy is an excellent and non-invasive alternative to obtaining size distribution of aggregates in precipitation processes.
ARTICLE | doi:10.20944/preprints201705.0126.v1
Subject: Biology, Other Keywords: amyloid peptides; androgen receptor; nuclear receptor; aggregation; atomic force microscopy
Online: 16 May 2017 (17:48:54 CEST)
The human androgen receptor (AR) is a ligand inducible transcription factor harboring an amino terminal domain (AR-NTD) hosting the ligand independent activation function. AR-NTD is intrinsically disordered and display aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence of 22 residues. The length of the polyQ sequence, as well as the presence of adjacent sequence motifs modulate this aggregation property. AR-NTD contains also a conserved sequence motif KELCKAVSVSM that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions of its conserved cysteine residue. As peptide sequences with intrinsic ability to oligomerize are reported to have an impact on the aggregation of polyQ tract, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR NTD, using Atomic Force Microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR NTD.
ARTICLE | doi:10.20944/preprints202301.0500.v1
Subject: Life Sciences, Other Keywords: Alzheimer’s Disease (AD) mitochondria; endoplasmic reticulum (ER); serial block-face scanning electron microscopy (SBFSEM); three-dimensional electron microscopy (3D EM); small molecule mitochondria targeted therapeutics
Online: 27 January 2023 (10:08:59 CET)
Alzheimer’s Disease (AD) has no cure. Earlier, we showed that partial inhibition of mitochondrial complex I (MCI) with small molecule CP2 induces adaptive stress response activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD. Here, using serial block-face scanning electron microscopy (SBFSEM) and three-dimensional (3D) EM reconstructions combined with Western blot analysis and next-generation RNA sequencing, we demonstrate that CP2 treatment also restores mitochondrial morphology and mitochondria-endoplasmic reticulum (ER) communication in the APP/PS1 mouse brain. Using 3D EM volume reconstructions, we show that mitochondria in AD dendrites exist primarily as mitochondria-on-a-string (MOAS). Compared to other morphological phenotypes, MOAS are extensively enveloped in the ER membranes forming multiple mitochondria-ER contact sites (MERCS) known to contribute to abnormal lipid and calcium homeostasis. CP2 treatment specifically reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reduction in MERCS, ER stress, and improved lipid homeostasis. These data provide novel information on the role MOAS play in AD and additional support for further development of partial MCI inhibitors as disease modifying strategy for AD.
COMMUNICATION | doi:10.20944/preprints202205.0087.v1
Subject: Materials Science, Surfaces, Coatings & Films Keywords: supramolecular aggregation of nanoparticles; aluminum and gold supports; scanning electron microscopy
Online: 7 May 2022 (03:19:17 CEST)
The supramolecular aggregation processes occurring on metallic (aluminum and gold) surfaces in aqueous solutions of bovine serum albumin (BSA) during drying were studied using advanced scanning electron microscopy (SEM). The possible mechanism for the formation of amazing intricate fractal structures on metallic surfaces was proposed based on the analysis of SEM images, size distribution diagrams and EDX-scanning elements distribution maps.
Subject: Materials Science, Biomaterials Keywords: Mining tailings; Tantalum; X-Ray diffraction; Raman spectroscopy; Scanning electron microscopy.
Online: 23 October 2020 (12:22:54 CEST)
In this work, a deep characterization of the properties of K6Ta10.8O30 microrods has been performed. The starting material used to grow the microrods has been recovered from mining tailings coming from the Penouta Sn-Ta-Nb deposit, located in the north of Spain. The recovered material has been submitted to a thermal treatment to grow the microrods. Then, they have been characterized by scanning electron microscopy, X-ray diffraction, micro-Raman and micro-photoluminescence. The results of our study confirm that the K6Ta10.8O30 microrods have a tetragonal tungsten bronze-like crystal structure, which can be useful for ion-batteries and photocatalysis.
ARTICLE | doi:10.20944/preprints202009.0255.v1
Subject: Chemistry, Food Chemistry Keywords: antioxidant enzymes; hydrogen peroxide; phenylalanine ammonia-lyase; proline; scanning electron microscopy
Online: 11 September 2020 (09:58:41 CEST)
Susceptibility of four blood orange cultivars (‘Moro’, ‘Tarocco’, ‘Sanguinello’ and ‘Sanguine’) to chilling injury (CI) was studied. Antioxidant enzymes, physiological and biochemical changes were measured monthly at 2 and 5 °C plus 2 days at 20 °C for shelf life. At 2 °C, CI symptoms were higher than at 5 °C, and ‘Moro’ and ‘Tarocco’ had significantly higher CI than ‘Sanguinello’ and ‘Sanguine’. ‘Moro’ and ‘Tarocco’ had the highest electrolyte leakage, malondialdehyde, H2O2 and polyphenol oxidase activity and lower phenylalanine ammonia-lyase compared with ‘Sanguinello’ and ‘Sanguine’. The scanning electron microscopy micrographs revealed that ‘Moro’ and ‘Taroco’ showed severe fractures in the flavedo due to CI. ‘Sanguinello’ and ‘Sanguine’ were more tolerant to CI due to an increase of catalase, ascorbate peroxidase and superoxide dismutase, which could prevent the loss of membrane integrity and alleviate CI symptoms. The order of susceptibility of cultivars to CI was ‘Moro’> ‘Tarocco’> ‘Sanguine’> ‘Sanguinello’.
Subject: Physical Sciences, Condensed Matter Physics Keywords: pulsed-laser deposition; in-situ x-ray diffraction; electron microscopy; multiferroics
Online: 4 December 2019 (12:28:04 CET)
Structure quality of LuFeO3 epitaxial layers grown by pulsed-laser deposition on sapphire substrates with and without platinum interlayers has been investigated by in-situ high-resolution x-ray diffraction (reciprocal-space mapping). The parameters of the structure such as size and misorientation of mosaic blocks have been determined as functions of the thickness of LuFeO3 during the PLD growth and for different platinum interlayers thicknesses up to 40 nm. The x-ray diffraction results combined with ex-situ scanning electron microscopy and high-resolution transmission electron microscopy demonstrate that the Pt interlayer significantly improves the structure of LuFeO3 by reducing the misfit of the LuFeO3 lattice with respect to the material underneath.
ARTICLE | doi:10.20944/preprints201809.0341.v1
Subject: Biology, Plant Sciences Keywords: Polar Auxin Transport; PIN transporters; Secretion; 3D-SIM microscopy; Mathematical modeling
Online: 18 September 2018 (10:09:24 CEST)
Intercellular transport of auxin is driven by PIN-formed (PIN) proteins. PINs are localized at the plasma membrane (PM) and on constitutively recycling endomembrane vesicles. Therefore, PINs can mediate auxin transport either by direct translocation across the PM or by pumping it into secretory vesicles (SVs), leading to its secretory release upon fusion with the PM. Which of these two mechanisms dominates is a matter of debate. Here we addressed the issue with a mathematical modeling approach. We demonstrate that the efficiency of secretory transport depends on SV size, half-life of PINs on the PM, pH, exocytosis frequency and PIN density. 3D-SIM microscopy was used to determine PIN density on the PM. Combing this data with published values of the other parameters, we show that the transport activity of PINs in SVs would have to be at least 1000x greater than on the PM in order to produce a comparable macroscopic auxin transport. If both transport mechanisms operated simultaneously and PINs were equally active on SVs and PM, the contribution of secretion to the total auxin flux would be negligible. In conclusion, while secretory vesicle-mediated transport of auxin is intriguing and theoretically possible model, it unlikely to be a major mechanism of auxin transport in planta.
ARTICLE | doi:10.20944/preprints201807.0248.v3
Subject: Chemistry, Other Keywords: ionic liquids, friction, lubrication mechanisms, surface forces apparatus, atomic force microscopy.
Online: 17 July 2018 (08:22:00 CEST)
The present study provides molecular insight into the mechanisms underlying energy dissipation and lubrication of a smooth contact lubricated by an ionic liquid. We have performed normal and lateral force measurements with a surface forces apparatus and by colloidal probe atomic force microscopy on following model systems: 1-ethyl-3-methyl imidazolium bis-(trifluoro-methylsulfonyl)imide, in dry state and in equilibrium with ambient (humid) air; the surface was either bare mica or functionalized with a polymer brush. The velocity-dependence of the friction force reveals two different regimes of lubrication, boundary-film lubrication, with distinct characteristics for each model system, and fluid-film lubrication above a transition velocity V*. The underlying mechanisms of energy dissipation are evaluated with molecular models for stress-activated slip and flow, respectively. The stress-activated slip assumes that two boundary layers (composed of ions/water strongly adsorbed to the surface) slide pass each; the bond dynamics and the strength of the interaction at the slip plane dictate the change in friction -decreasing, increasing or remaining constant- with velocity in the boundary-film lubrication regime. Above a transition velocity V*, friction monotonically increases with velocity in the three model systems. Here, layers of ions slide past each (“flow”) under a shear stress and friction depends on a shear-activation volume that is significantly affected by confinement. The proposed friction model provides a molecular perspective of the lubrication of smooth contacts by ionic liquids and allows identifying the physical parameters that control friction.
ARTICLE | doi:10.20944/preprints201702.0013.v1
Subject: Materials Science, Nanotechnology Keywords: nano-cube; heterostructures; bottom-up growth; electron microscopy; bandgap; Ag-WO3
Online: 4 February 2017 (09:23:00 CET)
A new class of nano-cube core-shell heterostructures containing Ag coating on the top of WO3 was fabricated. Physical vapor deposition was used to produce WO3 based nano-heterostructures. All kind of wet toxic chemical process was avoided to make the process simple and contaminant free. Sputtering of WO3 and a subsequent thermal annealing process was done to create nano-cubes of WO3. After that, sputtering of Ag was performed to form the Ag-WO3 core-shell nano-heterostructures (CSNH). The CSNHs were characterized using field emission scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS), X-ray diffraction analysis (XRD) and UV-vis spectroscopy. The morphologies, elemental analysis, interfaces, crystallinity, phases, and chemical compositions were analyzed. The bottom-up growth of WO3 nanocubes was studied using different time periods at 900°C. Ag coating was also studied before and after annealing. Finally, an optical property (band gap) was also analyzed using Tauc plot derive from absorption spectra. The tailoring the band gap of WO3 from ~2.9eV to ~ 2.45 eV was observed while Ag-WO3 CSNH formed.
ARTICLE | doi:10.20944/preprints202209.0024.v1
Subject: Life Sciences, Virology Keywords: flavivirus; tick-borne encephalitis; electron microscopy; EELS; nanoparticle tracking analysis; cryo-EM
Online: 1 September 2022 (10:54:41 CEST)
Tick-borne encephalitis virus (TBEV) is a RNA-containing enveloped virus, a member of the Flaviviridae family. Here we describe a detailed analysis of the size and structure of inactivated TBEV (the Sofyin-Chumakov TBEV strain, used in vaccines). Four analytical methods were used to analyze individual TBEV particles—negative staining TEM, cryo-EM, AFM, and NTA. All methods confirmed that the particles were monodisperse, and their mean size was ~50 nm. Cryo-EM data were used to obtain a 3D electron density model of the virus with clearly distinguishable E-proteins. STEM-EELS analysis detected phosphorous in the particles, which was interpreted as the RNA signal. Altogether, the described analytical procedures can be valuable for the further analysis of vaccine inactivated virus samples.
ARTICLE | doi:10.20944/preprints202112.0500.v1
Subject: Life Sciences, Biophysics Keywords: immunoglobulin G; complement component C1; high-speed atomic force microscopy; CH1; CL
Online: 31 December 2021 (10:36:38 CET)
Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component, C1q. To provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for quantitative visualization of the interaction between IgG and the C1 complex composed of C1q, C1r, and C1s. Results showed that C1q in the C1 complex is restricted regarding internal motion and has a stronger binding affinity for on-membrane IgG assemblages than C1q alone, presumably because of smaller conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG variant that lacks the entire CH1 domain in the absence of antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.
ARTICLE | doi:10.20944/preprints202112.0049.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: bacterial wilt; biological control; phage; microscopy; sequencing; molecular characterization; genomic characterization; depolymerase
Online: 3 December 2021 (10:36:47 CET)
Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plant were isolated from river water in Europe but not fully characterized, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum.
REVIEW | doi:10.20944/preprints202111.0091.v1
Subject: Biology, Other Keywords: microtubule organizing center; centrosome; centriolar plaque; Plasmodium; Toxoplasma gondii; ultrastructure expansion microscopy
Online: 3 November 2021 (16:35:39 CET)
Microtubule organizing centers (MTOCs) perform critical cellular tasks by nucleating, stabilizing and anchoring microtubule’s minus ends. These capacities impact tremendously a wide array of cellular functions ranging from ascribing cell shape to orchestrating cell division and generating motile structures, among others. The phylum Apicomplexa comprises over 6000 single-celled obligate intracellular parasitic species. Many of the apicomplexan are well known pathogens such as Toxoplasma gondii and the Plasmodium species, causative agents of toxoplasmosis and malaria, respectively. Microtubule organization in these parasites is critical for organizing the cortical cytoskeleton, enabling host cell penetration and the positioning of large organelles, drive cell division and direct the formation of flagella in sexual life stages. Apicomplexans are a prime example of MTOC diversity displaying multiple functional and structural MTOCs combinations within a single species. This diversity can only be fully understood in light of each organism's specific MT nucleation requirements and their evolutionary history. Insight into apicomplexan MTOCs had traditionally been limited to classical ultrastructural work by transmission electron microscopy. However, in the past few years, a large body of molecular insight has emerged. In this work we describe the latest insights into nuclear MTOC biology in two major human and animal disease causing Apicomplexans; Toxoplasma gondii and Plasmodium spp.
ARTICLE | doi:10.20944/preprints202105.0222.v1
Subject: Physical Sciences, Acoustics Keywords: magnetic force microscopy; calibration; reference samples; micromagnetism; metrology for magnetism; magnetic Multilayers
Online: 10 May 2021 (16:06:46 CEST)
Magnetic force microscopy (MFM) is a widespread technique for imaging magnetic structures with a resolution of some 10 nanometers. MFM can be calibrated to obtain quantitative (qMFM) spatially resolved magnetization data in units of A/m by determining the calibrated point spread function of the instrument, its instrument calibration function (ICF), from a measurement of a well-known reference sample. Beyond quantifying the MFM data, a deconvolution of the MFM image data with the ICF also corrects the smearing caused by the finite width of the MFM tip stray field distribution. However, the quality of the calibration depends critically on the calculability of the magnetization distribution of the reference sample. Here, we discuss a Ti/Pt/Co multilayer stack which shows a stripe domain pattern as a suitable reference material. A precise control of the fabrication process combined with a characterization of the sample micromagnetic parameters allows to reliably calculate the sample’s magnetic stray field, proven by a very good agreement between micromagnetic simulations and qMFM measurements. A calibrated qMFM measurement using the Ti/Pt/Co stack as a reference sample is shown and validated and the application area for quantitative MFM measurements calibrated with the Ti/Pt/Co stack is discussed.
Subject: Materials Science, Biomaterials Keywords: Microscopy Image Segmentation; Deep Learning; Data Augmentation; Synthetic Training Data; Parametric Models
Online: 1 March 2021 (13:07:00 CET)
The analysis of microscopy images has always been an important yet time consuming process in in materials science. Convolutional Neural Networks (CNNs) have been very successfully used for a number of tasks, such as image segmentation. However, training a CNN requires a large amount of hand annotated data, which can be a problem for material science data. We present a procedure to generate synthetic data based on ad-hoc parametric data modelling for enhancing generalization of trained neural network models. Especially for situations where it is not possible to gather a lot of data, such an approach is beneficial and may enable to train a neural network reasonably. Furthermore, we show that targeted data generation by adaptively sampling the parameter space of the generative models gives superior results compared to generating random data points.
ARTICLE | doi:10.20944/preprints202102.0183.v1
Subject: Physical Sciences, Acoustics Keywords: electrostatic force microscopy; proton exhange membrane; ionic domain; surface charge density; PEMFC
Online: 8 February 2021 (10:42:15 CET)
Understanding the ionic channel network of proton exchange membranes, which dictate fuel cell performance, is crucial when developing proton exchange membrane fuel cells. However, itis difficult to characterize due to complicated nano structure and differing changes to their structure with different amounts of water uptake. Electrostatic force microscopy (EFM) can map surface charge distribution as nano special resolution by measuring the electrostatic force between a vibrating conductive tip and a charged surface under an applied voltage, . In this study, the ionic channel network of a proton exchange membrane is analyzed using EFM. A mathematical approximation model of the ionic channel network is first derived, to explain changes in force gradient on the surface using EFM. The phase lag of dry and wet Nafion under stepwise changes to bias voltage is then measured. Based on the model, variations in the ionic channel network of Nafion with different amounts of water uptake are analyzed numerically. The mean surface charge density of both membranes, which is connected with the ionic channel network, is calculated using the model. The results show that the difference between the mean surface charge of the dry and wet membranes is consistent with the variation in their proton conductivity.
REVIEW | doi:10.20944/preprints202102.0163.v1
Subject: Life Sciences, Biochemistry Keywords: ATP; OXPHOS; ATP synthase; mitochondria; biochemical assays; fluorescence microscopy; genetically-encoded biosensors
Online: 5 February 2021 (14:45:04 CET)
ATP is the most universal and essential energy molecule in the eukaryotic cell. This is due to its ability to store energy in form of high energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, signalling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolysed with a spatiotemporal resolution is necessary to understand its functions both in physiological and pathological contexts. In this review, we will first describe the ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths and weaknesses. Then, we will explore the palette of genetically-encoded biosensors designed for microscope-based approaches and show how their spatiotemporal resolution opened up the possibility to follow ATP levels and production in living cells. Finally, we will comment on how ATP monitoring is used in preclinical practices, and to what extent genetically-encoded sensors could be used as a promising tool to elucidate pathologies in which ATP is implicated.
ARTICLE | doi:10.20944/preprints202101.0164.v1
Subject: Life Sciences, Biochemistry Keywords: Arbutus unedo L. Artificial Pollination, Breeding, Drought stress, Microscopy, Pollen, Physiological performance
Online: 8 January 2021 (14:08:49 CET)
Arbutus unedo L. is a small Ericaceae tree with a circum-Mediterranean distribution. It has a huge ecological impact on southern Europe forests and a great economic importance, as a source of phytochemicals with bioactive properties and for fruit production. On the foreseen climate change context, breeding towards drought tolerance is necessary in order to ameliorate plant performance. The aim of this work was therefore to study the reproduction mechanisms of strawberry tree, obtain new genetic combinations by hybridization and select genotypes more tolerant to drought stress. A morphological analysis of flowers and pollen was carried out, and controlled pollinations performed both in vitro and ex vitro. The very first approach on strawberry tree breeding by means of hybridization is also presented. Several physiological parameters were evaluated on 26 genotypes submitted to a water deficit regime. Plant behavior under drought greatly varied among genotypes, which showed a high phenotype plasticity. Three genotypes that were able to cope with water restriction without compromising net CO2 assimilation were identified as highly tolerant to drought stress. The results obtained elucidate the reproduction mechanisms of strawberry tree and open the way for a long-term breeding program based on the selection of drought tolerant plants.
ARTICLE | doi:10.20944/preprints201902.0004.v2
Subject: Life Sciences, Cell & Developmental Biology Keywords: artificial intelligence; machine learning; live-cell imaging; super-resolution microscopy; classification; segmentation
Online: 19 February 2019 (12:20:04 CET)
Artificial Intelligence based on Deep Learning is opening new horizons in Biomedical research and promises to revolutionize the Microscopy field. Slowly, it now transitions from the hands of experts in Computer Sciences to researchers in Cell Biology. Here, we introduce recent developments in Deep Learning applied to Microscopy, in a manner accessible to non-experts. We overview its concepts, capabilities and limitations, presenting applications in image segmentation, classification and restoration. We discuss how Deep Learning shows an outstanding potential to push the limits of Microscopy, enhancing resolution, signal and information content in acquired data. Its pitfalls are carefully discussed, as well as the future directions expected in this field.
REVIEW | doi:10.20944/preprints201812.0290.v1
Subject: Materials Science, Nanotechnology Keywords: Scanning tunneling microscopy; unimolecular electronics; molecular device; monolayer; coordination chemistry; interfaces; nanotechnology
Online: 24 December 2018 (15:39:00 CET)
The development of organic devices has been focused in their miniaturization in order to obtain denser and faster electronic circuits. The challenge is to build the devices adding atom by atom or molecule by molecule until the desired structure is achieved. To do this job, techniques able to see and manipulate matter at this scale are needed. Scanning tunneling microscopy has been the selected technique by scientists to develop smart and functional unimolecular devices. This review article compiles the latest developments in this field giving examples of supramolecular systems monitored and fabricated at molecular scale by bottom-up approaches using STM at solid/liquid interface.
ARTICLE | doi:10.20944/preprints201810.0603.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: super-resolution microscopy; quantum dots; cannabinoid receptor type 1; neuronal plasticity; synapses
Online: 25 October 2018 (11:19:17 CEST)
Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here we tested the possibility of extracting from the nano-resolved (16 ms and 30 nm) population dynamics of several quantum dots, time-binned at the second time-scale, the rapid structural changes of the cell membrane surface. We used for this proof-of-concept study bright, small and stable biofunctional QD nanoconstructs recognizing the neuronal cannabinoid type 1 (CB1) receptor and a commercial point-localization microscope to reconstruct in 3D the dynamics of the plasma membrane surface of cultured cells with a spatial resolution of tens of nanometers. CB1 receptor was chosen because it’s a highly expressed and fast diffusing membrane protein. Therefore, rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allowed highly precise reconstruction of the membrane surface in less than one minute. QD nanoconstructs diffused into the membrane of synaptic clefts allowing the entire topological reconstruction of the presynaptic component. In addition, we demonstrated successful reconstruction of the remarkably high dynamics of membrane surface topology at the second time-scale both in HEK-293 cell filopodia and axons. Our results show that this novel technique, which we named nanoPaint, is a powerful precision tool for the study of the structural plasticity of cell membrane surfaces.
ARTICLE | doi:10.20944/preprints201808.0323.v1
Subject: Materials Science, Nanotechnology Keywords: bimetallic nanoparticles; electron microscopy; molecular dynamics simulation; Gupta potential; PdPt alloys; nanostructures
Online: 18 August 2018 (05:59:06 CEST)
Bimetallic nanoparticles are of interest since they lead to many interesting electrical, chemical, catalytic, and optical properties. They are particularly important in the field of catalysis since they show superior catalytic properties than their monometallic counterparts. The structures of bimetallic nanoparticles depend mainly on the synthesis conditions and the miscibility of the two components. In this work, PdPt alloyed-bimetallic nanoparticles (NPs) were synthesized through the polyol method, and characterized using spherical aberration (Cs) corrected scanning transmission electron microscopy (STEM). High-angle annular dark-field (HAADF)-STEM images of bimetallic nanoparticles were obtained. The contrast of images shows that nanoparticles have an alloy structure with an average size of 8.2 nm. Together with the characterization of nanoparticles, a systematic molecular dynamics simulations study, focused on the structural stability and atomic surface segregation trends in 923-atom PdPt alloyed-bimetallic NPs was carried out.
REVIEW | doi:10.20944/preprints201803.0043.v2
Subject: Life Sciences, Biophysics Keywords: cryo-electron microscopy; air-water interface; conformational heterogeneity; focus gradient; radiation damage
Online: 3 May 2018 (11:08:20 CEST)
With forty years of developments, bio-macromolecule cryo-electron microscopy has met its revolution of resolution and is playing a very important role in structural biology study. According to different specimen states, cryo-electron microscopy (cryo-EM) involves three specific techniques, single particle analysis (SPA), electron tomography and sub-tomogram averaging, and electron diffraction. All these three techniques have not realized their full potentials of solving structures of bio-macromolecules and therefore need to be developed in the future. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which includes air-water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimen, and electron radiation damage. Besides, potential solutions of these bottlenecks are proposed and discussed, which are worthy of further investigations in the future.
ARTICLE | doi:10.20944/preprints201804.0112.v1
Subject: Engineering, Mechanical Engineering Keywords: atomic force microscopy; cantilever’s mathematical model; dynamic characteristics; nonlinear stiffness; high speed
Online: 10 April 2018 (07:39:25 CEST)
Increasing of the imaging rate of conventional atomic force microscopy (AFM) is almost impossible without impairing of the imaging quality, since the probe tip tends to lose contact with the sample. We propose to apply the additional nonlinear force on the upper surface of a cantilever, which will help to keep the tip and surface in contact. In practice this force can be produced by the precisely regulated airflow. Such an improvement affects the AFM system dynamics, which were evaluated using a mathematical model presented in this paper. The model defines the relationships between the additional nonlinear force, the pressure of the applied air stream and the initial air gap between the upper surface of the cantilever and the end of the air duct. It was found that the nonlinear force created by the stream of compressed air (aerodynamic force) prevents the contact loss caused by the high scanning speed or higher surface roughness, and at the same time has minimal influence on the interaction force, thus maintaining stable contact between the probe and the surface. This improvement allows to effectively increase the scanning speed by at least 10 times using a soft (spring constant of 0.2 N/m) cantilever by applying the air pressure of 40 Pa. If a stiff cantilever (spring constant of 40 N/m) is used, the potential of accuracy improvement reaches 92 times. This method is suitable for use with different types of AFM sensors and can be implemented practically without essential changes in AFM sensor design.
ARTICLE | doi:10.20944/preprints201608.0120.v1
Subject: Chemistry, Other Keywords: green nanotechnology; gold nanoparticles; biosynthesis; high resolution transmission electron microscopy; Cape flora
Online: 11 August 2016 (09:53:19 CEST)
The preparation of gold nanoparticles (AuNPs) involves a variety of chemical and physical methods. These methods use toxic and environmentally harmful chemicals. Consequently, the synthesis of AuNPs using green chemistry has been under investigation to develop eco-friendly nanoparticles. One method to achieve this is the use of plant-derived phytochemicals capable of reducing gold ions to produce AuNPs. The aim of this study was to implement a facile microtitre-plate method to screen a large number of aqueous plant extracts to determine the optimum concentration (OC) to bio-synthesize the AuNPs. Several AuNPs of different sizes and shapes were successfully synthesized and characterized from seventeen South African plants. The characterization was done using Ultra Violet-Visible Spectroscopy, Dynamic Light Scattering, High Resolution Transmission Electron Microscopy and Energy-Dispersive X-ray Spectroscopy. We also studied the effects of temperature on the synthesis of the nanoparticles and measured its effect on the particle size of the synthesized AuNPs and the data showed that changes in temperatures affect the size and dispersity of the generated AuNPs. Further, some of the synthesized AuNPs were stable upon incubation with different biological solutions in vitro.
ARTICLE | doi:10.20944/preprints202102.0545.v1
Subject: Life Sciences, Biochemistry Keywords: trastuzumab, HER2, extracellular vesicles, breast cancer, proteomic analysis, immunoelectron microscopy, TOP1, CD63, mitochondria.
Online: 24 February 2021 (11:38:41 CET)
Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is a ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance, and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Tz induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. As these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.
Subject: Life Sciences, Biophysics Keywords: cytosolic phospholipase A2; electron microscopy; C2 domain; Golgi; membrane bending; membrane curvature; oligomerization
Online: 12 March 2020 (04:10:52 CET)
Group IV phospholipase A2α (cPLA2α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA2α to the Golgi involves the N-terminal C2 domain whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA2α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigate how membrane curvature influences the homeostatic role of cPLA2α in vesicular trafficking. The cPLA2α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA2α membrane penetration. We show that cPLA2α undergoes C2 domain-dependent oligomerization on membranes in vitro and in A549 cells. We found that the association of the cPLA2α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization was observed on vesicles ~50 nm in diameter. We demonstrated that the cPLA2α C2 domain generates cholesterol enriched Golgi-derived vesicles independently of cPLA2α catalytic activity. Our results therefore provide novel insight into the molecular forces that mediate C2 domain-dependent membrane localization in vitro and in cells.
ARTICLE | doi:10.20944/preprints201905.0003.v1
Subject: Materials Science, Metallurgy Keywords: Shape memory alloys; CuZr alloy; Thermal cycling; Microstructure; Martensitic transformation; Transmission electron microscopy
Online: 3 May 2019 (14:10:58 CEST)
Equiatomic CuZr alloy undergoes a martensitic transformation from the B2 parent phase to martensitic phases (P21/m and Cm) below 150 °C. We clarified the effect of the thermal cycling on the morphology and crystallography of martensite in equiatomic CuZr alloy using a transmission electron microscopy. The 10th cycled specimens consisted of different multiple structures at the maximum temperature of DSC measurement: 400 °C and 500°C, respectively. At the maximum temperature 400 °C of DSC measurement, it is composed of the fine plate-like variants, and a lamellar eutectoid structure consisting of Cu10Zr7 and CuZr2 phases on the martensitic variant. Concerning the maximum temperature 500 °C of DSC measurement, it is observed the martensitic structure and the lamellar structure in which the martensitic phase was completely eutectoid transformed. The formation of this lamellar eutectoid structure due to thermal cycling leads to the shift of forward and reverse transformation peaks to low and high temperature side. In addition, new forward and reverse transformation peaks indicating a new transformation appeared by thermal cycling, and the peaks remained around -20 °C. This new martensitic transformation behavior is also discussed.
ARTICLE | doi:10.20944/preprints201709.0093.v1
Subject: Materials Science, Surfaces, Coatings & Films Keywords: CAD/CAM implant abutments; zirconia; surface roughness; soft tissue adhesion; focus variation microscopy
Online: 20 September 2017 (05:28:24 CEST)
Objective: CAD/CAM generated ceramic implant abutments have recently attracted interest due to their superior customization possibilities and aesthetic advantages. Despite their widespread clinical use, little information is currently available on their surface topography, however. The transmucosal portion of the abutment shoulder is of particular interest, as it ideally supports soft tissue but minimizes mechanical plaque retention. The aim of this in vitro study was to topographically characterize the trans- and subgingival roughness of CAD/CAM zirconia abutments from different manufacturers and compare them with zirconia stock abutments. Material and Method: The surface topography of eight CAD/CAM zirconia implant abutments (tests) and two prefabricated zirconia stock abutments (controls) was determined using focus variation microscopy. Two points on the abutment shoulder were subjected to profilometric examination. 2D and 3D parameters of roughness were obtained and compared. Results: The surface roughness of all the test abutments exceeded the recommended threshold of Ra = 0.2 µm and therefore exhibited an increased risk of mechanical plaque retention. Obvious differences in surface structure were apparent, allowing conclusions to be drawn about the manufacturing method and subsequent reworking processes. Conclusion: Manually reworking the trans- and submucosal area of the investigated CAD/CAM zirconia abutments appears necessary to fulfil the conditions for optimal surface topography. The Sa value as arithmetic mean, taking the maximum height (Sz value) and surface excess (Sdr) into account, is an essential parameter for assessing the surface topography of implant abutments.
ARTICLE | doi:10.20944/preprints201703.0212.v1
Subject: Materials Science, Biomaterials Keywords: hydroxyapatite; xenografts; scanning electron microscopy; degradation; resorption; Ca/P ratio; bone response; biocompatibility
Online: 28 March 2017 (17:09:44 CEST)
Some studies have demonstrated that in vivo degradation processes are influenced by the material’s physico-chemical properties. The present study compares two hydroxyapatites manufactured on an industrial scale, deproteinized at low and high temperatures, and how physico-chemical properties can influence the mineral degradation process of material performance in bone biopsies retrieved 6 months after maxillary sinus augmentation. Residual biomaterial particles were examined by field scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) to determine the composition and degree of degradation of the bone graft substitute material. According to the EDX analysis, the Ca/P ratio significantly lowered in the residual biomaterial (1.08±0.32) compared to the initial composition (2.22±0.08) for the low-temperature sintered group, which also presented high porosity, low crystallinity, low density, a large surface area, and poor stability and a high resorption rate compared to the high-temperature sintered material. This demonstrates that variations in the physico-chemical properties of bone substitute material clearly influence the degradation process. Further studies are needed to determine whether the resorption of deproteinized bone particles proceeds slowly enough to allow sufficient time for bone maturation to occur.
COMMUNICATION | doi:10.20944/preprints202210.0466.v1
Subject: Materials Science, Polymers & Plastics Keywords: Polymer matrix composites; POSS; Fracture mechanics; J-integral; Stress intensity factor; Scanning electron microscopy
Online: 31 October 2022 (06:19:34 CET)
Polyhedral oligomeric silsesquioxane (POSS) is a suitable nanoscale reinforcement for thermosetting polymers, such as epoxy resin systems in order to modify its mechanical, thermal, and chemical properties. The inclusion of POSS in the epoxy resin at higher loading (greater than 1 wt.%), however, introduces the ductility during the fracture behavior of these nanocomposites. Consequently, the J-integral is used to quantify the fracture behavior of these materials and characterize the crack growth resistance curve against stable crack growth. A range of nanocomposites is prepared by adding 0.5, 1, 3, 5, and 8 wt.% of glycidyl POSS into DGEBF epoxy resin cured with an amine-based curing agent. From the fracture toughness experiment the load-displacement result confirms that when the POSS reinforcement is greater than 1 wt.%, the fracture behavior of the nanocomposite changes from brittle to ductile. For both brittle and ductile nanocomposites, the addition of POSS molecules improves the crack initiation toughness. The development of POSS–POSS compliant domains are reported previously, is attributed for this change in the failure behavior. The fractured images of POSS–epoxy nanocomposites, obtained using scanning electron microscopy, show that the increase in fracture resistance at higher values of POSS loading occurs due to the extensive shear yielding. Meanwhile, the increased fracture toughness at lower values of POSS loading occurs due to crack pinning and crack deflection.
ARTICLE | doi:10.20944/preprints202204.0312.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: Tensional homeostasis; Traction microscopy; Gastric cancer cells; E-cadherin mutations; Extra-cellular matrix proteins
Online: 30 April 2022 (05:18:13 CEST)
In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we investigated the effect of E-cadherin mutations affecting distinct protein domains on tensional homeostasis of gastric cancer cells. We used micropattern traction microscopy to measure temporal fluctuations of cellular traction forces in AGS cells transfected with the wild-type E-cadherin or with variants affecting the extracellular, the juxtamembrane, and the intracellular domains of the protein. We focused on the dynamic aspect of tensional homeostasis, namely the ability of cells to maintain a consistent level of tension, with low temporal variability around a set point. Cells were cultured on hydrogels micropatterned with different extracellular matrix (ECM) proteins to test whether the ECM adhesion impacts cell behavior. A combination of Fibronectin and Vitronectin was used as a substrate that promotes the adhesive ability of E-cadherin dysfunctional cells, whereas Collagen VI was used to test an unfavorable ECM condition. Our results showed that mutations affecting distinct E-cadherin domains influenced differently cell tensional homeostasis, and pinpointed the juxtamembrane and intracellular regions of E-cadherin as the key players in this process. Furthermore, Fibronectin and Vitronectin might modulate cancer cell behavior towards tensional homeostasis.
ARTICLE | doi:10.20944/preprints202202.0317.v1
Subject: Materials Science, Nanotechnology Keywords: photochromic; diarylethene; bipyridine; high-resolution scanning tunneling microscopy; cooperative switches; molecular orbital; DFT calculations
Online: 24 February 2022 (13:17:29 CET)
Diarylethene is a prototypical molecular switch that can be reversibly photoisomerized between its open and closed forms. Ligands bpy-DAE-bpy, consisting of a phenyl-diarylethene-phenyl (DAE) central core and bipyridine (bpy) terminal substituents, are able to self-organize. They are investigated by scanning tunneling microscopy at the solid-liquid interface. Upon light irradiation, cooperative photochromic switching of the ligands is recognized down to the sub-molecular level. The closed isomers show different electron density of states (DOS) contrasts, attributed to the HOMO or LUMO molecular orbitals observed. More importantly, the LUMO images show remarkable differences between the open and closed isomers, attributed to combined topographic and electronic contrasts mainly on the DAE moieties. The electronic contrasts from multiple HOMO or LUMO distributions, combined with topographic distortion of the open or closed DAE, are interpreted by density functional theory (DFT) calculations.
ARTICLE | doi:10.20944/preprints202104.0259.v1
Subject: Materials Science, Biomaterials Keywords: VAR alloy obtaining; Biomedical applications; Apatite films; Corrosion, X-ray method, Electron microscopy; Hardness
Online: 9 April 2021 (13:24:09 CEST)
In this paper, a novel biocompatible alloy defined as FeMoTaTiZr was obtained and functionalized by hydroxyapatite-based coatings (HAP) in order to increase their biocompatibility, bioactivity, and resistance to corrosion for to be used as bone implants. To obtain the surface with antibacterial properties, the HAP coatings were doped with small amount of Zn. The alloy was prepared using the VAR (Vacuum Arc Remelting) equipment, while the coatings by RF magnetron sputtering method. The EDS analysis confirmed the presence of Ca and P in the case of all developed coatings, having Ca/P or Ca/(P+Zn) ratio of about 1.70 and 1.66, respectively. The XRD and ATR-FTIR investigations confirmed the presence of calcium phosphate phases. The roughness of uncoated substrates increased after coating with HAP, and it was considerably increased by the Zn addition. The electrochemical tests showed that the un-doped HAP exhibited good corrosion behavior, while Zn doped HAP coatings have a high dissolution rate in fetal bovine serum, being more proper as a biodegradable material.
TECHNICAL NOTE | doi:10.20944/preprints202101.0034.v1
Subject: Biology, Plant Sciences Keywords: clearing; 3D imaging; Arabidopsis thaliana; embryo; confocal microscopy; cell wall staining; fluorescent reporter; GFP
Online: 4 January 2021 (12:34:36 CET)
Tissue clearing methods combined with confocal microscopy have been widely used for studying developmental biology. In plants, ClearSee is a reliable clearing method that is applicable to a wide range of tissues and is suitable for gene expression analysis using fluorescent reporters, but its application to the Arabidopsis thaliana embryo, a model system to study morphogenesis and pattern formation, has not been described in the original literature. Here we describe a ClearSee-based clearing protocol, which is suitable for obtaining 3D images of Arabidopsis thaliana embryos. The method consists of embryo dissection, fixation, washing, clearing, and cell wall staining, and enables high quality 3D imaging of embryo morphology and expression of a fluorescent reporter with the cellular resolution.
ARTICLE | doi:10.20944/preprints201911.0224.v1
Subject: Materials Science, Metallurgy Keywords: Cu6Sn5 intermetallic; solid-state polymorphic transition; superstructure; transmission electron microscopy; damage mechanism; multiple reflows
Online: 19 November 2019 (04:05:13 CET)
The formation of high-melting-point Cu6Sn5 interconnections is crucial to overcome the collapse of Sn-based micro-bumps and produce reliable intermetallic interconnections in three-dimensional (3D) package. However, because of the multiple reflows in 3D package manufacturing, Cu6Sn5 interconnections will experience the cyclic polymorphic transitions in the solid state. The repeated and abrupt change in the Cu6Sn5 lattice due to the cyclic polymorphic transitions can cause extreme strain oscillations, producing damages at the surface and in the interior of the Cu6Sn5 matrix. Moreover, because of the polymorphic-transition-induced grain splitting and superstructure phase formation, the reliability of Cu6Sn5 interconnections will thus face great challenges in 3D package. In addition, the Cu6Sn5 polymorphic transition is structure-dependent, and the η′↔η polymorphic transition will occur at the surface while the η′↔ηs↔η polymorphic transition will occur in the deep matrix. Our results can provide in-depth understandings of structural evolution and damage mechanism of Cu6Sn5 interconnections in real 3D package manufacturing.
ARTICLE | doi:10.20944/preprints201906.0251.v1
Subject: Physical Sciences, Applied Physics Keywords: video microscopy, imaging, automated data acquisition, nanoparticle tracking, measurement embedded applications, open-source software
Online: 25 June 2019 (12:53:50 CEST)
We introduce PyNTA, a modular instrumentation software for live particle tracking. By using the multiprocessing library of Python and the distributed messaging library pyZMQ, PyNTA allows users to acquire images from a camera at close to maximum readout bandwidth while simultaneously performing computations on each image on a separate processing unit. This publisher/subscriber pattern generates a small overhead and leverages the multi-core capabilities of modern computers. We demonstrate capabilities of the PyNTA package on the featured application of nanoparticle tracking analysis. Real-time particle tracking on megapixel images at a rate of 50 Hz is presented. Reliable live tracking reduces the required storage capacity for particle tracking measurements by a factor of approximately 103, as compared with raw data storage, allowing for a virtually unlimited duration of measurements
CASE REPORT | doi:10.20944/preprints201803.0211.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: MTC; calcitonin; parafollicular C cells; secretory granules; immunofluorescence; ultrastructure; transmission electron microscopy; ERBB1; ERBB2
Online: 26 March 2018 (09:05:18 CEST)
Medullary thyroid carcinomas (MTCs) are rare thyroid tumors occurring in both sporadic and hereditary forms and whose pathogenesis is related to RET proto-oncogene alterations. MTCs originate from parafollicular cells, which produce calcitonin that represents the biochemical activity of MTC. Total thyroidectomy is the main treatment for MTC and often cures patients with confined diseases. In cases of metastasis, the approach depends on the rate of progression of disease. We report a case of a 54 years old female with a single, incidentally discovered, thyroid nodule of 1 cm, classified as suspicious MTC after a stimulation test with i.v. calcium. After surgery, we examined the nodule using immunohistochemistry, immunofluorescence and electron microscopy. In addition to calcitonin, we found that it expressed intracellular positivity for the RTK receptors ERBB1 and ERBB2. Consistently with MTC features, ultrastructural examination of the tumor displayed heterogeneous spindle-shaped cells containing two groups of secretory granules. Due to the significant correlation found between high ERBB1/ERBB2 levels in MTCs and extrathyroidal growth, the detection of ERBB1 and ERBB2 expression suggests that the two oncoproteins may possibly be involved in tumor proliferative responses and/or differentiation of C-cells. The biological, prognostic and therapeutic significance of these patterns would merits further investigations.
ARTICLE | doi:10.20944/preprints201803.0165.v1
Subject: Materials Science, Nanotechnology Keywords: elastomers; lattice model; Monte Carlo simulation; surface tensions; small angle scattering; transmission electron microscopy
Online: 19 March 2018 (16:04:52 CET)
The properties of rubber are strongly influenced by the distribution of filler within the polymer matrix. Here we introduce a Monte Carlo-based morphology generator. The basic elements of our model are cubic cells, which, in the current version, can be either silica filler particles or rubber volume elements in adjustable proportion. The model allows the assignment of surface free energies to the particles according to whether a surface represents, for instance, 'naked' silica or silanised silica. The amount of silanisation is variable. We use a nearest-neighbour site-exchange Monte Carlo algorithm to generate filler morphologies, mimicking flocculation. Transmission electron micrographs (TEM) as well as small angle scattering (SAS) intensities can be calculated along the Monte Carlo trajectory. In this work we demonstrate the application of our morphology generator in terms of selected examples. We illustrate its potential as a tool for screening studies, relating interface tensions between the components to filler network structure as characterized by TEM and SAS.
ARTICLE | doi:10.20944/preprints202301.0238.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: induced pluripotent stem cells; embryoid bodies; light-sheet microscopy; multicolor fluorescence in situ hybridization; chromosomes
Online: 13 January 2023 (07:10:46 CET)
Embryoid bodies (EBs) are multicellular three-dimensional (3D) aggregates generated from induced pluripotent stem cells (iPSCs) in suspension and serve as useful biological sources for many downstream applications. Imaging of live EBs has been hampered mainly due to the inherent limitations of the imaging techniques applied to date. This study aimed to image human iPSC (hiPSC) derived EBs to obtain their 3D volume, determining size, morphology, and cell viability from day 7 to 14 using Light Sheet Fluorescence Microscopy (LSFM). Furthermore, chromosomal stability was assessed using Multicolor fluorescence in situ hybridization (M-FISH) from day 8 to 14. EB volume increased from day 7 to 13 which, decreased at day 14. From day 7 to 11, the EBs mainly appeared spherical and morphed into an ellipsoidal shape by day 13. All EBs showed varied external morphologies and larger cavities at day 14. The EB karyotype was diploid 46XY at day 8 and exhibited a low level of aneuploidy from day 10 to 14. This study shows that an increase in cell death affects the morphology and chromosomal stability in EBs derived from hiPSC. We demonstrate that the combination of LSFM and M-FISH helps characterize EBs that will assist future stem cell therapies.
ARTICLE | doi:10.20944/preprints202204.0109.v1
Subject: Physical Sciences, Optics Keywords: self-design setup; real-time imaging; GPU acceleration; quantitative phase imaging; differential phase contrast microscopy
Online: 12 April 2022 (10:19:06 CEST)
Quantitative differential phase contrast (qDPC) imaging has become an important method of optical measurement and life science research in microscopy because of its high reconstruction resolution and non-invasive, high-contrast and quantitative imaging of biological samples. Despite the continuous development of the principle and algorithm, the frame rate of the existing qDPC algorithm is still much lower than that of camera acquisition, so it is hardly applied to real-time image the fast-moving biological samples. In this paper, based on color-coded multiplexing strategy, a compact real-time quantitative phase imaging system is designed to realize multi-mode imaging. The system employs a programmable LED array to illuminate directly, and the phase reconstruction algorithm is deployed in the graphics processing unit (GPU) of the laptop to accelerate the calculation. The system can achieve high-speed quantitative phase imaging of non-stained biological samples, and the frame rate can reach 60fps. The device has the advantages of compact structure, low cost and portability. Thus, it is suitable for mobile medical applications.
ARTICLE | doi:10.20944/preprints202107.0084.v1
Subject: Physical Sciences, Other Keywords: Ferritin; Quantum dots; Layer-by-layer deposition; Conductive atomic force microscopy; Strong correlations; mott insulator
Online: 5 July 2021 (10:07:03 CEST)
Highly-correlated electrons – electrons that engage in strong electron-electron interactions – have been observed in transition metal oxides and quantum dots and can create unusual material behavior that is difficult to model, such as switching between a low resistance metal state and a high resistance Mott insulator state. Tests of devices using a layer-by-layer deposition process for forming multilayer arrays of ferritin (a transition metal (iron) oxide storage protein) have been previously reported that indicate that highly-correlated electron transport is occurring, consistent with models of electron transport in quantum dots. This paper reports the results of the effect of various degrees of structural homogeneity on the electrical characteristics of these ferritin arrays, as well as demonstrating that these structures can provide a switching function associated with the circuit that they are contained within, consistent with the observed behavior of highly-correlated electrons.
Subject: Medicine & Pharmacology, Allergology Keywords: gas chromatography mass spectrometry; metabolomics; acute lymphoblastic leukemia; confocal microscopy; high-throughput drug screens; VAST
Online: 7 June 2021 (08:01:21 CEST)
Transplant of human cancer cells into zebrafish larvae has emerged as a useful methodology in cancer research. Zebrafish have very low husbandry costs, are amenable to large-scale drug screening, and are unmatched for optical clarity in live animal imaging. However, there is currently no consensus on the ideal methods for xenograft of human cancer cells into zebrafish. Here, we have examined the effects of transplant site and housing temperature on both zebrafish larvae and human cancer cells using survival analyses, metabolomic approaches, and in vivo imaging. Our data show that while zebrafish larvae can adapt to the ideal conditions for mammalian cells, human cancer cells are highly sensitive to both temperature change and transplant site. Human cells housed in slightly cooler than physiologic temperatures had a significantly altered metabolism that resulted in changes in growth, survival, and response to chemotherapy. Cancer cells xenografted into the yolk of the larvae also had reduced proliferation and drug response compared to those xenografted into the soma, in part due to the differences in metabolites available at these sites. In total, temperature and transplant site can have profound effects on xenografted cells. Standardization of zebrafish xenograft methods will enhance data reproducibility between individual laboratories.
ARTICLE | doi:10.20944/preprints202011.0360.v1
Subject: Life Sciences, Biochemistry Keywords: Riboflavin transporter deficiency; motor neurons; mitochondria; energy metabolism; electron microscopy; antioxidants; SOD2; oxidative stress; neurodegeneration
Online: 12 November 2020 (18:14:17 CET)
Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated iPSC lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells dramatically impacting cell redox status. In the present work, we extend our study to motor neurons (MNs), i.e., the cell type mostly affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis, following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker, and βIII Tubulin, as neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by Focused Ion Beam/Scanning Electron Microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient specific RTD models, and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs, emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies, aimed at correcting mitochondrial defects, based on the use of antioxidants.
ARTICLE | doi:10.20944/preprints202001.0269.v1
Subject: Materials Science, Metallurgy Keywords: Niobium-titanium microalloyed steel; Electrical resistivity; Atom probe tomography; Solute niobium; Scanning transmission electron microscopy
Online: 23 January 2020 (16:15:44 CET)
Microalloying of low carbon steel with niobium (Nb) and titanium (Ti) is standardly applied in high-strength low-alloy (HSLA) steels enabling austenite conditioning during thermo-mechanical controlled processing (TMCP), which results in pronounced grain refinement in the finished steel. The metallurgical effects of microalloying elements are related solute drag and precipitate particle pinning, both acting on the austenite grain boundary thereby delaying or suppressing recrystallization of the deformed grain. In that respect it is important to better understand the precipitation kinetics as well as the precipitation sequence in a typical Nb-Ti-microalloyed steel. Various characterization methods have been utilized in this study for tracing microalloy precipitation after simulating different austenite TMCP conditions in a Gleeble apparatus. Atom probe tomography (APT), scanning transmission electron microscopy in a focused ion beam equipped scanning electron microscope (STEM-on-FIB) and electrical resistivity measurements provide complementary information on the precipitation status and are correlated with each other. It will be demonstrated that accurate electrical resistivity measurements can monitor the general consumption of solute microalloys (Nb) during hot working which was complemented by APT measurements of the steel matrix. On the other hand, STEM revealed that a large part of Nb-containing particles during hot working are co-precipitated with titanium during cooling from the austenitizing temperature. Precipitates that form during cooling or isothermal holding can be distinguished from strain-induced precipitates by corroborating STEM measurements with APT results. APT specifically allows obtaining detailed information about the chemical composition of precipitates as well as the distribution of elements inside the particle. Electrical resistivity measurement, on the contrary, provides macroscopic information on the progress of precipitation and can be calibrated by APT. The current paper highlights the complementarity of these methods and shows first results within the framework of a larger study on strain-induced precipitation.
ARTICLE | doi:10.20944/preprints201902.0082.v1
Subject: Life Sciences, Biochemistry Keywords: positional isomerism; PUFA biosynthesis; membrane remodelling; membrane lipidomics; lipidomic analysis; fluidity; functional two photon microscopy
Online: 8 February 2019 (09:43:06 CET)
Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional MUFA isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 μM) and evidence the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodeling was influenced by the type of fatty acid and positional isomer, with increase of 8cis-18:1, n-10 PUFA and decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in case of sapienic acid. EC50 of sapienic acid (232.3 μM at 96 hrs) was the highest found among the tested fatty acids, thus influencing cell viability that was only reduced at 25% at 300 μM, whereas palmitoleic acid induced cell death. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.
ARTICLE | doi:10.20944/preprints201811.0492.v1
Subject: Chemistry, Electrochemistry Keywords: single atom catalysis; carbon-supported catalysts; platinum-group metals; aberration-corrected scanning transmission electron microscopy
Online: 20 November 2018 (09:19:06 CET)
Nanoparticles of platinum-group metals (PGM) on carbon supports are widely used as catalysts for a number of chemical and electrochemical conversions on laboratory and industrial scale. The newly emerging field of single atom catalysis focuses on the ultimate level of metal dispersion, i.e. atomically dispersed metal species anchored on the substrate surface. However, the presence of single atoms in traditional nanoparticle-based catalysts remains largely overlooked. In this work we use aberration-corrected scanning transmission electron microscope to investigate four commercially available nanoparticle-based PGM/C catalysts (PGM = Ru, Rh, Pd, Pt). We show that in addition to nanoparticles, single atoms are also present on the surface of carbon substrates. These observations raise questions about the role that single atoms play in conventional nanoparticle PGM/C catalysts. We critically discuss the observations with regard to the quickly developing field of single atom catalysis.
ARTICLE | doi:10.20944/preprints201804.0094.v1
Subject: Materials Science, Other Keywords: surface; textiles; flame retardant; plasma; ultraviolet; durability; phosphorus; nitrogen; polyurethane; thermal analysis; scanning electron microscopy
Online: 8 April 2018 (11:59:49 CEST)
Conventional flame retardant (FR) application processes for textiles involve aqueous processing which is resource intensive in terms of energy and water usage. Recent research using sol-gel and layer-by-layer chemistries, while claimed to be based on more environmentally-sustainable chemistry, still require aqueous media with the continuing problem of water management and drying processes being required. This paper outlines the initial forensic work to characterise commercially produced viscose/flax, cellulosic furnishing fabrics which have had conferred upon them durable flame retardant (FR) treatments using a novel, patented atmospheric plasma/UV excimer laser facility for processing textiles with the formal name - Multiplexed Laser Surface Enhancement (MLSE) system. This system (MTIX Ltd., UK), is claimed to offer the means of directly bonding of flame retardant precursor species to the component fibres introduced either before plasma/UV exposure or into the plasma/UV reaction zone itself, thereby eliminating a number of wet processing cycles. Nine commercial fabrics, pre-impregnated with a semi-durable, proprietary FR finish and subjected to the MLSE process have been analysed for their flame retardant properties before and after a 40 °C 30 min water soak. For one fabric, the pre-impregnated fabric was subjected to a normal heat cure treatment which conferred the same level of durability as the plasma/UV-treated analogue. TGA and LOI were used to further characterise their burning behaviour and the effect of the treatment on surface fibre morphologies were assessed. Scanning electron microscopy indicated that negligible changes had occurred to surface topography of the viscose fibres occurred during plasma/UV excimer processing.
CASE REPORT | doi:10.20944/preprints201710.0116.v1
Subject: Materials Science, Biomaterials Keywords: biomineralization; calcium enriched material; calcium deficient hydroxyapatite; dentinal tubule; energy dispersive spectroscopy; scanning electron microscopy
Online: 17 October 2017 (11:50:20 CEST)
This case report describes evidence of intratubular biomineralization in root canal filled with calcium enriched material after 8 years of clinical maintenance. The schematic findings of dentinal tubules were investigated with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The root canal obturation material was closely adapted to root dentin surface, suggesting the possibility of chemical bonding between the two interfaces. SEM and EDS observation of dentinal tubules showed intratubular biomineralized crystal structures with Ca/P ratio in a range of 1.30–2.12, suggesting bioactive capacity of calcium enriched cement.
TECHNICAL NOTE | doi:10.20944/preprints202103.0030.v1
Subject: Biology, Anatomy & Morphology Keywords: Marine invertebrates; RNA extraction by LiCl precipitation; electron microscopy of blood cells; cytology stains; tunicate culture
Online: 1 March 2021 (14:19:14 CET)
Marine invertebrates are model organisms in several areas of biological sciences, being a source of massive biological information. Although, the scientific relevance of marine invertebrates, the research with them can be limited for their tissue characteristics and troubles for the replication of physical and chemical properties of seawater. Thence, the main goal of this laboratory workflow is to provide a useful methodological approach to reduce the experimental limitations during the study of marine invertebrates. The present study describes experimental methodologies for the collection, transport, and maintenance of sessile tunicates. Also, an approach to observe and characterize, a diverse population of blood cells in marine invertebrates, by several cytological stains and electron microscopy. Lastly, suggestions and protocols to extract quality RNA from samples with high concentrations of salts, pigments, secondary metabolites, and polysaccharides. This methodological approach can be easily adapted to other marine invertebrates, moreover uses low-cost reagents and widely available laboratory equipment. Making possible the study of different types of marine animals in diverse locations.
ARTICLE | doi:10.20944/preprints202008.0184.v2
Subject: Earth Sciences, Environmental Sciences Keywords: particulate pollution; air quality; deposition flux; geostatistics; ordinary kriging; urban-industrial; elemental identification; electronic scanning microscopy
Online: 11 September 2020 (06:01:45 CEST)
This work aimed to assess and characterize the air quality in what concerns particulate pollution in urban-industrial environments. The main objectives were to study the spatial variability of the deposition flux of particulate pollution identifying areas with higher deposition, associate the variability with climatological variables and with possible surrounding emitting sources. The method for collecting the deposited particles was based on the standard NF X 43-007. Sampling for particulate pollution took place between April 2015 and February 2016, through seven sampling campaigns. Maps of the spatial dispersion for the particulate pollution were obtained through statistics and geostatistics techniques. Elemental identification by scanning electron microscopy (SEM) was also used but only in two sampling campaigns. The results show that the sampling campaigns that took place during hot and dry periods, 2nd and 3rd, present higher deposition flux: 2.04 g/(m2 x month) and 1.72 g/(m2 x month), respectively. Lower deposition fluxes were registered in the 6th and 7th campaigns: 0.23 g/(m2 x month) and 0.24 g/(m2 x month), respectively. It was also observed a recurrent high deposition at specific sampling points which may be due to both the nearby road traffic and the presence of chimneys. SEM analysis allowed to associate repetitive element deposition, at the same sampling point, to the same emitting source.
ARTICLE | doi:10.20944/preprints201912.0267.v1
Subject: Chemistry, Electrochemistry Keywords: adsorption; coatings; poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate); corrosion tests; atomic force microscopy
Online: 20 December 2019 (07:00:55 CET)
Poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) named further PVBA was investigated as protective coating for copper corrosion in 0.9 % NaCl solution using electrochemical measurements such as, electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization associated with Atomic Force Microscopy (AFM). The PVBA coating on the copper surface (Cu-PVBA) was modeled in methanol containing PVBA. Its inhibitory properties against corrosion was comparatively discussed with those of the copper sample treated in methanol without polymer (Cu-Me) and of untreated sample (standard copper). A protective performance of PVBA coating of 80 % was computed from electrochemical measurements, for copper corrosion in NaCl solution. Also, AFM images designed a specific surface morphology of coated surface with PVBA, clearly highlighting a polymer film adsorbed on the copper surface, which presents certain deterioration after corrosion, but metal surface was not significantly affected compared to those of untreated samples or treated in methanol, in the absence of PVBA.
ARTICLE | doi:10.20944/preprints201704.0010.v1
Subject: Materials Science, Polymers & Plastics Keywords: PLA; fused deposition modeling (FDM); surface characterization; vibrational spectroscopy; laser confocal microscopy; X-ray photoelectron spectroscopy
Online: 3 April 2017 (17:34:40 CEST)
Polylactic Acid (PLA) is an organic polymer commonly used in fused deposition (FDM) printing and biomedical scaffolding that is biocompatible and immunologically inert. However, variations in source material quality and chemistry make it necessary to characterize the filament and determine potential changes in chemistry occurring as a result of the FDM process. We used several spectroscopic techniques, including laser confocal microscopy, Fourier-Transform Infrared (FTIR) spectroscopy and photoacousitc FTIR spectroscopy, Raman spectroscopy, and X-ray photoelectron Spectroscopy (XPS) in order to characterize both the bulk and surface chemistry of the source material and printed samples. Scanning Electron Microscopy (SEM) and Differential Scanning Calorimetry (DSC) were used to characterize morphology, crystallinity, and the glass transition temperature following printing. Analysis revealed calcium carbonate-based additives which were reacted with organic ligands and potentially trace metal impurities, both before and following printing. These additives became concentrated in voids in the printed structure. This finding is important for biomedical applications as carbonate will impact subsequent cell growth on printed tissue scaffolds. Results of chemical analysis also provided evidence of the hygroscopic nature of the source material and oxidation of the printed surface, and SEM imaging revealed micro and sub-micron scale roughness that will also impact potential applications.