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Quality of extracellular vesicle images by transmission electron microscopy is operator and protocol dependent
摘要： Transmission electron microscopy (TEM) has nanometre resolution and can be used to distinguish single extracellular vesicles (EVs) from non-EV particles. TEM images of EVs are a result of operator image selection. To which extent operator image selection reflects the overall sample quality, and to which extent the images are comparable and reproducible, is unclear. In a first attempt to improve the comparability and reproducibility of TEM to visualise EVs, we compared operator image selection to images taken at predefined locations from the same grids, using four EV TEM preparation protocols, a single EV-containing sample and a single TEM instrument. Operator image selection leads to high-quality images that are more similar between the protocols. In contrast, images taken at predefined locations reveal differences between the protocols, for example in number of EVs per image and background quality. From the evaluated protocols, for only one protocol the operator image selection is comparable to the TEM images taken at predefined locations. Taken together, operator image selection can be used to demonstrate the presence of EVs in a sample, but seem less suitable to demonstrate the quality of a sample. Because images taken at predefined locations reflect the overall quality of the EV-containing sample rather than the presence of EVs alone, this is a first step to improve the comparability and reproducibility of TEM for monitoring the quality of EV-containing samples.
关键词： urine,negative staining,transmission electron microscopy (TEM),Exosomes,extracellular vesicles (EVs),microvesicles
更新于2025-09-23 15:23:52
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Night-Time Oxidation of a Monolayer Model for the Air–Water Interface of Marine Aerosols—A Study by Simultaneous Neutron Reflectometry and in Situ Infra-Red Reflection Absorption Spectroscopy (IRRAS)
摘要： This paper describes experiments on the ageing of a monolayer model for the air–water interface of marine aerosols composed of a typical glycolipid, galactocerebroside (GCB). Lipopolysaccharides have been observed in marine aerosols, and GCB is used as a proxy for these more complex lipopolysaccharides. GCB monolayers are investigated as pure films, as mixed films with palmitic acid, which is abundant in marine aerosols and forms a stable attractively mixed film with GCB, particularly with divalent salts present in the subphase, and as mixed films with palmitoleic acid, an unsaturated analogue of palmitic acid. Such mixed films are more realistic models of atmospheric aerosols than simpler single-component systems. Neutron reflectometry (NR) has been combined in situ with Fourier transform infra-red reflection absorption spectroscopy (IRRAS) in a pioneering analysis and reaction setup designed by us specifically to study mixed organic monolayers at the air–water interface. The two techniques in combination allow for more sophisticated observation of multi-component monolayers than has previously been possible. The structure at the air–water interface was also investigated by complementary Brewster angle microscopy (BAM). This study looks specifically at the oxidation of the organic films by nitrate radicals (NO3?), the key atmospheric oxidant present at night. We conclude that NO3? oxidation cannot fully remove a cerebroside monolayer from the surface on atmospherically relevant timescales, leaving its saturated tail at the interface. This is true for pure and salt water subphases, as well as for single- and two-component films. The behaviour of the unsaturated tail section of the molecule is more variable and is affected by interactions with co-deposited species. Most surprisingly, we found that the presence of CaCl2 in the subphase extends the lifetime of the unsaturated tail substantially—a new explanation for longer residence times of materials in the atmosphere compared to lifetimes based on laboratory studies of simplified model systems. It is thus likely that aerosols produced from the sea-surface microlayer at night will remain covered in surfactant molecules on atmospherically relevant timescales with impact on the droplet’s surface tension and on the transport of chemical species across the air–water interface.
关键词： palmitic acid,monolayer,neutron,nitrate radical,palmitoleic acid,reflectivity,aerosol,infra-red,cerebroside,Brewster angle microscopy
更新于2025-09-23 15:23:52
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Signal enhancement in laser diode thermal desorption-triple quadrupole mass spectrometry analysis using microwell surface coatings
摘要： Laser diode thermal desorption (LDTD) is an ionization source usually coupled to triple quadrupole mass spectrometry (QqQMS) and specifically designed for laboratories requiring high-throughput analysis. It has been observed that surface coatings on LDTD microwell plates can improve the sensitivity of the analysis of small polar molecules. The objective of the present study is to understand and quantify the effect of microwell surface coatings on signal intensity of small organic molecules of clinical, environmental and forensic interest. Experiments showed that the peak areas of diclofenac, chloramphenicol, salicylic acid and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol obtained by LDTD-QqQMS increased by up to 3 orders of magnitude when using microwells coated with ethylenediaminetetraacetic acid (EDTA). Tests with different chelating agents and polytetrafluoroethylene as microwell surface coatings showed that nitrilotriacetic acid gave significantly higher peak areas for five out of the nine compounds that showed signal enhancement using chelating agents as coatings. Scanning electron microscopy studies of EDTA-coated and uncoated microwells showed that analytes deposited in the former formed more uniform and thinner films than in the latter. The enhancement effect of surface coatings in LDTD-QqQMS was explained mainly by the formation of homogenous and thinner layers of nanocrystals of analytes that are easier to desorb thermally than the layers formed when the analytes dry in direct contact with the bare stainless steel surface. Chemisorption of some analytes to the stainless steel surface of the microwell plate appeared to be a minor factor. Surface coatings widen the number of compounds analyzable by LDTD-QqQMS and can also improve sensitivity and limits of detection.
关键词： pharmaceuticals,high-throughput,hormones,pesticides,x-ray photoelectron spectroscopy,scanning electron microscopy,stainless steel,small organic molecules,chelating agents
更新于2025-09-23 15:23:52
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Hyperspectral holographic microscopy of bio-objects based on a modified Linnik interferometer
摘要： We present a modified Linnik microinterferometer that can register image-plane hyperspectral holograms in reflection of various microobjects in incoherent light. A voice coil was integrated into the reference arm of the interferometer to enable mirror displacement. A set of interferograms is registered in each pixel of a high-speed CMOS camera. Post processing yields spectrally resolved image-plane holograms of the object. Quantitative phase images of red blood cells are obtained for each spectral component σ = 1/λ.
关键词： microscopy,Linnik interferometer,holography,white light interferometry,Fourier transform microspectroscopy
更新于2025-09-23 15:23:52
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Applications of nanostructures in wide-field, label-free super resolution microscopy
摘要： Super resolution imaging capable of resolving details beyond the diffraction limit is highly desired in many scientific and application fields, including bio-medicine, nanomaterial science, and opto-electronic integration. Up to now, many different methods have been proposed, among which wide-field, label-free super resolution microscopy is indispensable due to its good applicability to diverse sample types, large field of view (FOV), and high imaging speed. In recent years, nanostructures have made a crucial contribution to the wide-field, label-free subdiffraction microscopy, with various working mechanisms and configuration designs. This review summarizes the recent applications of the nanostructures in the wide-field, label-free super resolution microscopy, with the emphasis on the designs of hyperlens with hyperbolic dispersion, microsphere with “nano-jets”, and nanowire ring illumination microscopy based on spatial frequency shift effect. The bottlenecks of the current techniques and possible solutions are also discussed.
关键词： spatial resolution,nanowire,metamaterials,optical microscopy
更新于2025-09-23 15:23:52
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Experimental Observation of Bound States of 2D Dirac Electrons at Surface Steps of the Topological Insulator Bi <sub/>2</sub> Se <sub/>3</sub>
摘要： Topologically protected surface states of three-dimensional topological insulators provide a model framework for studying massless Dirac electrons in two dimensions. Usually a step on the surface of a topological insulator is treated as a scatterer for the Dirac electrons, and the study of its effect is focused on the interference of the incident and scattered electrons. Then a major role plays the warping of the Dirac cone far from the Dirac point. The existence of another significant effect near the Dirac point brought about by the presence of steps is experimentally demonstrated here. Namely the band bending in the vicinity of steps leads to formation of 1D bound states in the corresponding potential wells. The observation of bound states in such potential wells in our scanning tunneling microscopy and spectroscopy investigation of the surface of the topological insulator Bi2Se3 is reported. Numerical simulations support our conclusion and provide a recipe for the identification of such states.
关键词： density of states,Dirac electrons,surface states,edge states,bound states,scanning tunneling microscopy,Bi2Se3,topological insulators,scanning tunneling spectroscopy
更新于2025-09-23 15:23:52
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Analysis and Comparison of Tissue-Marking Dye Detection via Light Microscopy, Telemicroscopy, and Virtual Microscopy
摘要： Objectives: To examine the fidelity of ink color identification using light microscopy (LM), telemicroscopy (TM), and virtual microscopy (VM). Methods: Twenty H&E-stained frozen section slides, prepared after tissue inking with five stain combinations, were assessed by three pathologists using LM, TM, and VM. TM was performed using Mikroscan D2 slide scanner/LiveQ software with various objectives. VM was performed using Mikroscan D2 scanner/Qumulus software, specimens digitized at 20×. Results: Sensitivity/specificity by LM was 100%/100% for all colors. TM showed high overall specificity but poor sensitivity, particularly red (54%). VM showed high specificity for all colors except black (69%) and, consequently, poor sensitivity for all colors except black (96%). Conclusions: TMD identification via telepathology showed loss of sensitivity/specificity vs LM and highlighted the need for caution when interpreting TMDs with digital technologies and the need for validation protocols.
关键词： Virtual microscopy,Tissue-marking dye,Telepathology,Telemicroscopy
更新于2025-09-23 15:23:52
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Development of novel correlative light and electron microscopy linkage system using silicon nitride film
摘要： In this study, we investigated the optical properties of a silicon nitride (SiN) film. The thin SiN film (30 nm thick) exhibited good light transmittance and little autofluorescence and could be used as a microscope slide for optical microscopy (OM). In addition, we developed a novel correlative light and electron microscopy (CLEM) that combines OM with transmission electron microscopy (TEM) using an SiN thin film. In this system, CLEM was performed by replacing a detachable retainer with a holder for TEM and an adaptor for OM. The advantage of this method is that the same specimens can be sequentially observed using suitable OM and TEM.
关键词： TEM holder,correlative microscopy,SiN film,exchangeable retainer,OM–TEM integration
更新于2025-09-23 15:23:52
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Direct Imaging of Surface Plasmon-Driven Hot Electron Flux on the Au Nanoprism/TiO <sub/>2</sub>
摘要： Direct measurement of hot electron flux from a plasmonic Schottky nanodiode is important for obtaining fundamental insights explaining the mechanism for electronic excitation on a surface. Here, we report the measurement of photo-induced hot electrons on a triangular Au nanoprism on TiO2 under incident light with photoconductive atomic force microscopy (pc-AFM), which is direct proof of the intrinsic relation between hot electrons and localized surface plasmon resonance. We find that the local photocurrent measured on the boundary of the Au nanoprism is higher than that inside the Au nanoprism, indicating that field confinement at the boundary of the Au nanoprism acts as a hot spot, leading to the enhancement of hot electron flow at the boundary. Under incident illumination with a wavelength near the absorption peak (645 nm) of a single Au nanoprism, localized surface plasmon resonance resulted in the generation of a higher photo-induced hot electron flow for the Au nanoprism/TiO2, compared with that at a wavelength of 532 nm. We show that the application of a reverse bias results in a higher photocurrent for the Au nanoprism/TiO2, which is associated with a lowering of the Schottky barrier height caused by the image force. These nanoscale measurements of hot electron flux with pc-AFM indicate efficient photon energy transfer mediated by surface plasmons in hot electron-based energy conversion.
关键词： hot electron,Photoconductive atomic force microscopy,localized surface plasmon resonance,Schottky diode,field confinement,Au nanoprism
更新于2025-09-23 15:23:52
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Method for Combined Observation of Serial Sections of Stented Arteries Embedded in Resin by Light Microscopy and Transmission Electron Microscopy
摘要： We have developed a new method for obtaining information on whole tissues by light microscopy (LM) and ultrastructural features by transmission electron microscopy (TEM). This method uses serial sections of a stented artery embedded in resin. Stents were implanted in porcine coronary arteries in this study. The heart was perfusion fixed in a 2% paraformaldehyde and 1.25% glutaraldehyde mixed solution. The stented artery was then removed, fixed in 1% osmium, embedded in Quetol 651 resin, and sectioned serially. For LM, the black color of osmium was removed from the section by immersion in periodic acid and hydrogen peroxide after deplasticization. These sections were stained with hematoxylin and eosin and Elastica–Masson trichrome stain. For TEM, thin sections were re-embedded in Quetol 812 resin by the resupinate method and cut into ultrathin sections. A clear, fine structure was obtained, and organelles, microvilli, and cell junctions in the endothelium were easily observed. The combined observation of adjacent specimens by LM and TEM enabled us to relate histopathological changes in the millimeter scale to those in the nanometer scale.
关键词： porcine,stent,resupinate method,coronary artery,epoxy resin,transmission electron microscopy,light microscopy
更新于2025-09-23 15:22:29