Confocal Scanning Optical Microscope Research Articles

Far-Field Super-Resolution Optical Microscopy for Nanostructures in a Reflective Substrate

The resolution of an optical microscope is determined by the overall point spread function of the system. When examining structures significantly smaller than the wavelength of light, the contribution of the background or surrounding environment can profoundly affect the point spread function. This research delves into the impact of reflective planar substrate structures on the system’s resolution. We establish a comprehensive forward imaging model for a reflection-type confocal laser scanning optical microscope, incorporating vector field manipulation to image densely packed nanoparticle clusters. Both theoretical and experimental findings indicate that the substrate causes an interference effect between the background field and the scattered field from the nanoparticles, markedly enhancing the overall spatial resolution. The integration of vector field manipulation with an interferometric scattering approach results in superior spatial resolution for imaging isolated particles and densely distributed nanoscale particle clusters even with deep subwavelength gaps as small as 20 nm between them. However, the method still struggles to resolve nanoparticles positioned directly next to each other without any gap, necessitating further work to enhance the resolving ability. This may involve techniques like deconvolution or machine learning-based post-processing methods.

Preparation and Characterization of CuCrO2 Prepared by Solid State Reaction Method

AbstractRecently, delafossite materials have captured the interest of numerous researchers due to their unique characteristics of p‐type conductivity and transparency. These materials exhibit promising potential in various applications such as photovoltaics, sensors, and transparent electronics. This paper presents a detailed account of the preparation of CuCrO2 delafossite via the solid‐state reaction method. The structural properties of the sample are analyzed using XRD which confirms rhombohedral crystal structure with R‐3m space group. The optical band gap was determined to be 3.39 eV through UV‐visible spectroscopy. The morphology is studied using Laser Confocal scanning optical microscopy and the estimated grain size is found to be within the range of 7 µm.

The use of confocal microscopy and SEM for the determination of cut marks on human bones. Its application to a case study, in the foothill of Jujuy, Argentina (2000-1500 AP)

This article presents results from the analysis of a human skull worked by cuts, from an archaeological site in the San Francisco Valley, Northwestern Argentina (2000-1500 AP). The reassembly of the fragments allowed for the reconstruction of the skull shape and revealed this important perimortem modification. Although numerous individuals with signs of pre-burial interventions have been recorded in the region, this finding is unique because of the kind of work done on the skull.Three microscopic analysis techniques were employed in order to understand the type of instrument used in this modification and to reconstruct the technical sequence of the work process, including: observation with stereoscopic magnifying glass, confocal scanning optical microscopy, and SEM. Different analyses with high magnification technology allowed us to recognize various macro and micro marks that were the product of the operations carried out when working the bone. The results indicate the use of different tools, and establish the steps that intervened in the modification of the skull. Finally, these findings are discussed within the framework of the regional archaeological context.

EVALUATION OF WOOD SURFACE ROUGHNESS BY CONFOCAL MICROSCOPY

The main aim of this study is to define the usability of the confocal scanning optical microscope (CSOM) to evaluate the wood surface roughness. Therefore, systematic investigation was carried out to define the influences of CSOM on the acquisition of 2D surface roughness parameters. Mahr Perthometer was applied to get reference data to estimatetheapplicability of the CSOM. Because wood roughness parameters measured with stylus and optical methods are not always comparable a calibration method was conducted on a metal calibration etalon. After the calibration process, the roughness profiles taken with the optical and stylus units were much closer to each other and only the optical Rpkparameter was definitely higher due to artificial peaks generated by the optical system. In order to eliminate this measuringfailure, the morphological filter option of the optical apparatus may be activated. Thesurface roughness parameters were measured on planed Scotch pine samples. The planed surface plains were produced with 0.2 mm parallel offset to investigate thestructural influence of the single cutting plains. The obtained results show that the average values for Ra, Rq, Rz, Rk, and Rvkare close to each other for both measuring systems, only theoptically measured Rpkvalues must be corrected. The standard deviations, however, are systematically slightly higher for optical system. This may be explained by the higher resolution of the optical system giving more fine profile details. The earlier developed and introduced dimensionless quantities, such as Abbott ratio, are also fully comparable for both systems provided that the optically measured Rpkvalues are also correct.

Surface finishing effects on the corrosion behavior and electrochemical activity of 2098-T351 aluminum alloy investigated using scanning microelectrochemical techniques

The effects of surface finishing on the corrosion behavior and electrochemical activity of AA2098-T351 (Al–Cu–Li alloy) were investigated on the basis of the correlation between surface chemistry, microstructure and electrochemical activity. The alloy was evaluated in the as-received and polished conditions. The morphology of the two types of surfaces was investigated using confocal laser scanning microscopy (CLSM), optical microscopy and optical 3D profilometry. The surface chemistry was analyzed by X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDX). Scanning microelectrochemical techniques (namely, localized electrochemical impedance spectroscopy (LEIS), the scanning vibrating electrode technique (SVET) and scanning electrochemical microscopy (SECM) in potentiometric mode) were used to examine the electrochemical activity of the surfaces. The results showed that on the as-received surface, the near surface deformed layer (NSDL), which is composed of Mg-rich bands, influenced the corrosion activity of the alloy. Higher electrochemical activity and greater susceptibility to severe localized corrosion were related to the polished surface condition compared to the as-received one. • Surface finishing effects on chemical activity of 2098-T351 Al–Cu–Li alloy studied using scanning microelectrochemical techniques. • The effect of the near-surface deformed layer (NSDL) on 2098-T351 Al–Cu–Li was investigated for different surface finishes. • A solid contact magnesium ion-selective microelectrode (Mg 2+ ISME) employed to monitor Mg 2+ distributions over the surfaces. • SVET, LEIS, and SECM in potentiometric operation produced high spatial resolution images of reactive sites on the surfaces. • The surface chemistry, microstructure and electrochemical activity of the alloy with and without NSDL were correlated.

Binding of lambda carrageenan to bovine serum albumin and non-equilibrium effects of complexation

Binding of lambda carrageenan (lCG) to bovine serum albumin (BSA) and the effect of the formation path, namely mixing (M) at specific pH versus titration (T) from a neutral mixture, on complexation have been studied using turbidimetry, phase analysis, dynamic light scattering, confocal laser scanning and bright-field optical microscopy, circular dichroism and fluorescence measurements. The sizes and shape of the complex particles formed under T-conditions differ significantly from those obtained under M-conditions, and at maximal complexation T-complexes contain significantly less lCG as compared to M-complexes. At a pH below pIBSA phase analysis reveals two domains of q corresponding to an approximately constant lCG/BSA weight ratio in the complex phase q* (domain I), and a decreasing q* (domain II). Binding stoichiometries at pH 4.2 and 4.7 are 1:77 and 1:119 mole lCG/mole BSA. The yield of BSA in the complex phase is higher than 98% at pH 4.2 and 4.7, and the complexes form and are stable in the presence of 1.0M–1.5M NaCl. The interaction did not lead to protein denaturation or significant changes in the tertiary and secondary structure and illustrates an enhancement of the hydrophobicity of the protein within the complex particles. At pH 5.2 (pH on the wrong side of PIBSA) up to 37.6% BSA was bound to lCG, and the weight ratio of lCG/BSA in the complex particles (q*) was weakly dependent on q in the complete q range studied. The molecular origin of the unusually strong interaction between lCG and BSA is discussed.

Rapid absolute sizing of deeply subwavelength dielectric nanoparticles by confocal scanning optical microscopy

Accurate sizing of individual nanoparticles is crucial for the understanding of their physical and chemical properties and for their use in nanoscale devices. Optical sizing methods are non-invasive, rapid, and versatile. However, the low optical response of weakly absorbing subwavelength dielectric nanoparticles poses a fundamental challenge for their optical metrology. We demonstrate scalable optical sizing of such nanoparticles based on confocal scanning microscopy. The method is absolutely calibrated by correlating the optical signatures in the scattered pump laser signal to the ground truth nanoparticle sizes measured by an atomic force microscope. Using an air objective with a numerical aperture of 0.9, we measured the sizes of nanodiamond particles ranging from 35 to 175 nm, with an average error of ± 12.7 nm compared to the ground truth sizes. This technique paves the way for the metrology of a wide range of weakly scattering nano-objects for applications in biomedicine, catalysis, nanotechnology, and quantum optics.

Process parameters effect on high-temperature friction and galling characteristics of AA7075 sheets

ABSTRACT During hot forming of high-strength 7xxx series alloy, friction and galling often occur on the contact surface, degrade product surfaces quality, and affect severely by the process parameters, especially by load and speed. In the present paper, the effect of process parameters, namely applied load and sliding speed, on high-temperature friction and galling performance of AA7075 sheets has been determined using linear sliding tests at 350°C in simulated hot forming. The microscopic observations, i.e., scanning electron microscopy (SEM) and three-dimensional confocal scanning optical microscopy, were utilized to analyze hard phase distribution of the alloy as well as surface and sub-surface of the posttest specimens. A friction model was proposed to describe the friction mechanism. The results show that wear rate gradually decreased with increasing load and speed, attributing to the formed compacted oxide layer on the worn surface, which inhibited the rate of further attrition. Besides, for all test conditions, adhesion dominated the wear mechanism, and the compacted layer was damaged more serious due to more initiation of micro-cracks between hard phase and matrix and higher contact interface temperature at higher load and speed, thus the extent of adhesion is grievous resulting in a higher wear loss.

A chemical patterning approach of dense and porous titanium surfaces by using a combination of concentrated acid and oxidant

Commercially dense pure titanium sheets and porous titanium samples processed by powder metallurgy were treated with a mixture consisting of equal volumes of H2SO4 and H2O2 for 2 or 4 hours. Characterization was performed by scanning electron microscopy, energy dispersive X-ray spectroscopy, confocal scanning optical microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The analyses showed that the chemical patterning approach using a combination of concentrated acid and oxidant was able to generate a nanotexture on dense and porous titanium surfaces. In addition, the treated samples presented an oxide layer consisting predominantly of titanium dioxide with negative charge conferred by the presence of hydroxyl groups, which is an important factor that favors apatite nucleation and protein adsorption. It was also observed that oxide formation was more effective on porous samples than on dense samples, which can be explained by the higher surface area intrinsic to porous media. Finally, the findings indicated that both treatment times promoted similar modifications in surface properties, such as nanotexture and chemical composition, suggesting that the time of 2 hours were enough to induce the surface alterations at the nanoscale.

Fabrication and characterization of Pickering emulsion stabilized by soy protein isolate-chitosan nanoparticles

This work aimed to investigate the feasibility of fabricating food-grade Pickering emulsions stabilized by soy protein isolate-chitosan (SPI-CS) nanoparticles. The SPI-CS nanoparticles were characterized by scanning electron microscopy and particle size analyzer. Pickering emulsion stabilized by SPI-CS nanoparticles were characterized by confocal laser scanning microscopy and optical microscopy. The results showed that the average size of SPI-CS nanoparticles was 148.52 ± 1.95 nm. The droplet sizes of Pickering emulsions at different nanoparticle concentrations and oil fractions were 11.80 ± 2.20–43.68 ± 4.12 μm. SPI-CS nanoparticles stabilized Pickering emulsions at different nanoparticle concentrations and oil fractions had low creaming index, and these Pickering emulsions were stable at 4 °C for 20 days. These SPI-CS Pickering emulsions were stable at broad ionic strengths (0–1000 mM) and temperature (4–60 °C). These findings may provide new insight into the development of food-grade Pickering emulsions suitable for the formulation of functional foods.

Microstructure evolution of hot work tool steel 5CrNiMoV throughout heating, deformation and quenching

The microstructure evolutions of hot work tool steel 5CrNiMoV throughout the heating, deformation and quenching procedures are investigated. Microstructures of the heated, compressed, and cooled specimens are observed by high-temperature confocal laser scanning microscope (HT-CLSM), optical microscope, and electron backscatter diffraction (EBSD). The measurement shows that the austenite transformation starts at 730 °C and ends at 765 °C. After the temperature exceeds 850 °C during the heating, the in-situ observed grains are first refined by static recrystallization (SRX) and then grow with the holding time, accompanied by the grain boundary gradually become straight. The microstructures of hot compressed specimens indicate that the dynamic recrystallization (DRX) fraction increases with growing temperature and strain, in which discontinuous dynamic recrystallization (DDRX) is the main mechanism of DRX. The grain boundaries formed by DDRX are serrated and have a larger curvature, which are different from those formed by SRX and subsequent grain growth. The lath martensite generated from the water quenching following the compression tends to align parallel to each other in the prior austenite grain (PAG). It comprises the packets containing the elongated blocks with the same habit planes.

Real-space light-reflection mapping of atomically thin WSe2 flakes revealing the gradient local strain

The spatially continuous control of the physical properties in semiconductor materials is an important strategy in increasing electron-capturing or light-harvesting efficiencies, which is highly desirable for the application of optoelectronic devices including photodetectors, solar cells and biosensors. Unlike the multi-layer growth of chemical composition modulation, local strain offers a convenient way to continuously tune the physical properties of a single semiconductor layer, and open up new possibility for band engineering within the 2D plane. Here, we demonstrate that the gradient refractive index and bandgap can be generated in atomically thin transition metal dichalcogenide flakes due to the effect of thermal strain difference. A highly resolved confocal scanning optical microscopy is used to perform a real-space light-reflection mapping of suspended atomically thin WSe2 flakes at the low temperature of 4.2 K, in which the parabolic light-reflection profiles have been observed on suspended monolayer and bilayer WSe2 flakes. This finding is corroborated by our theoretical model which includes the effect of strain on both the refractive index and bandgap of nanostructures. The inhomogeneous local strain observed here will allow new device functionalities to be integrated within 2D layered materials, such as in-plane photodetectors and photovoltaic devices.

Coupling Effect of Prior Austenite Grain Size and Inclusion Characteristics on Acicular Ferrite Formation in Ti-Zr Deoxidized Low Carbon Steel

The coupling effect of prior austenite grain size and inclusion characteristics on acicular ferrite (AF) formation was investigated in Ti-Zr deoxidized low carbon steel by utilizing the high temperature confocal laser scanning microscope (HT-CLSM), optical microscope (OM), and scanning electron microscope (SEM) equipped with energy-dispersive spectrometer (EDS). The results indicated that with the target heating temperature increased from 1100 °C to 1350 °C, the average size of prior austenite grain varied from 58.22 to 237.40 μm, and the average grain size increased rapidly when the temperature was above 1200 °C. For inclusion characteristics, different target heating temperatures had no obvious effect on inclusion types, but had a great influence on the average size and number density of each, especially for the intragranular effective inclusions. In addition, as the increase of target heating temperature, the types of microstructure were identical, but both AF volume fraction and AF relative nucleation ability increased first and then decreased. When the target heating temperature of sample was 1250 °C, the AF volume fraction reached the maximum of 49.48 pct. However, the AF relative nucleation ability reached the maximum of 474.5 at the target heating temperature 1200 °C, at this time, the AF volume fraction was 47.92 pct, only 1.56 pct smaller than that at 1250 °C. Therefore, considering the AF volume fraction and AF relative nucleation ability, the optimal target heating temperature for AF formation in this study was 1200 °C, and the corresponding prior austenite grain size was 69.58 μm.

Release mechanisms of bioactive compounds in fruits submitted to high hydrostatic pressure: A dynamic microstructural analysis based on prickly pear cells.

High hydrostatic pressure (HHP) promotes the release of bioactive compounds from their intracellular compartments making them more bioaccessible. Our aim was to propose a schematic tissue model to explain the release mechanisms of betalains and phenolic compounds in vegetable cells submitted to HHP by analyzing cell microstructure, cell morphology, cell viability and the localization of bioactive compounds in prickly pear fruits. Prickly pear slices were pressurized at 100, 350 and 600MPa at 20°C. Chlorenchyma cells (in peels) and parenchyma cells (in pulps) were analyzed by transmission electron microscopy, confocal laser scanning microscopy and optical microscopy. After pressurization, the respiration and ethylene production of processed fruits were measured every 6h (during storage at 16°C and 75% RH for 24h). In chlorenchyma cells, HHP ruptured betalain-storing vesicles in the cytoplasm and possibly increased the activity of endogenous enzymes. Contrarily, HHP released betalains from the vacuoles of parenchyma cells due to breaking of the tonoplast where they presented higher stability. In both tissues, phenolic compounds were released from cell walls with increasing pressure and enhanced by cell wall ultrastructural modifications (100MPa), rupture (350MPa) and the rearrangement of microfibrillated cellulose (600MPa). Prickly pears submitted to HHP presented advanced senescence marked by considerable ethylene increase and the gradual loss of CO2 production after 6h. Cells were viable at 100MPa by conserving intact cell membranes and after 24h their respiration rates presented no significant differences compared to controls therefore indicating the possibility of synthesis of bioactive compound as a response to abiotic stress. We have proposed a new approach for analyzing the effects of HHP and have identified the storing of betalains in vesicles located in the cytoplasm of chlorenchyma cells for the first time. This study is the first to fathom the dynamic morphological changes and release mechanisms of bioactive compounds in vegetable cells subjected to HHP.

Emulsifying stability of enzymatically hydrolyzed egg yolk granules and structural analysis

Egg yolk granules (EYG) are natural and unique protein-lipid complexes, but their application in the food industry is limited because of their poor solubility and emulsifying properties. In this paper, the structural characteristics, emulsifying properties and interfacial adsorption were compared between the untreated granules (UG) and the enzymatically hydrolyzed granules (EG) (treated with subtilisin). EG-stabilized emulsion did not form cream at 0.2%–1.0% protein concentrations, while UG-stabilized emulsion formed a cream layer quickly after emulsification. The emulsion stability index of EG increased by nearly 8 times increased from 0.2% to 1.0%. The scanning electron microscope, confocal laser scanning microscopy (CLSM) and optical microscopy indicated that enzymatic hydrolysis changed the aggregation state of the EYG in powder, suspension and emulsions. Also, it was proven that enzymatic hydrolysis strengthened the internal hydrogen bonding and increased surface wettability and surface charge. The CLSM images confirmed that EYG are Pickering particles. The interfacial tension dropped to 8.4 mN/m after enzymatic hydrolysis. The SDS-PAGE analysis of the protein showed that enzymatic hydrolysis induced changes in the granules and interface, resulting in a strong adsorption capacity for EG at the interface. The enzymatic modifications of EYG promoted their emulsifying properties and showed that EYG have the potential to be used as novel food-grade particles through structural modification.

Using Confocal Laser Scanning Microscopy to Study Fossil Inclusion in Baltic Amber, a New Approach

We demonstrate that confocal laser scanning microscopy could be successfully used for rapid search of the organisms in the dark part of the amber samples. Combination of mid-infrared spectroscopy, confocal laser scanning microscopy and optical microscopy, allowed to identify and assess the quality of Baltic amber with fossil inclusion. Inclusion was identified as a spider from suborder Araneomorphae family Typhlopidae and it was struck by the mycelium.

Development of a confocal scanning microscope for fluorescence imaging and spectroscopy at variable temperatures.

We developed and tested a confocal scanning optical microscope that fits into a thermally controlled, commercial research cryostat designed for operation from ambient temperature down to below 4 K. The home-built microscope is a fiber-coupled, self-contained instrument based on readily available mechanical and optical components. Its sample module is sealed in a protective stainless steel tube that minimizes vibrations caused by the flow of cryogenic gas. A high numerical aperture microscope objective specifically designed for cryogenic and high-vacuum applications focuses the excitation light onto the sample, while the core of an optical fiber attached to an avalanche photodiode acts as the confocal detection pinhole. The sample is displaced using a piezotube scanner mounted on top of a three-axis, low-temperature nanopositioner assembly for coarse sample positioning. A broadband polarizing cube beam splitter in the emission path allows for polarization-resolved imaging and spectroscopy. Fluorescence excitation scans are acquired with custom-written software that correlates fluorescence photon counts with the output from a high precision wavelength meter, which is part of a narrow-band, tunable dye laser setup. The imaging and spectral data acquisition capabilities of the microscope were confirmed using a variety of samples and excitation wavelengths at temperatures ranging from 5 K to room temperature.

Evaluation of Lubrication Properties of Refortified Hydraulic Oils

Used lubricating oil contains more than 90% of good base oil and available additives, providing the potential of energy conservation, environmental protection, and economy, though it loses its useful performance because of contamination by impurities, oxidation, and degradation of additives. Many countries around the world have focused on reclamation and re-refining methods to treat and reuse waste lubricating oils for many years. But the regeneration rate of waste oils is very low in China, less than 20% of the lubricant consumption. Few studies on the performance, especially lubrication properties of recycled lubricating oils, have been considered. In this article, based on differences between used and fresh oils, used oils were reclaimed and replenished with additives (henceforth refortified oils). The physical and chemical properties, including viscosity, total acid number, moisture content, copper corrosion, and water separability from petroleum oil and synthetic fluid as well as the lubrication characteristics of three kinds of refortified hydraulic oils were investigated. The lubrication performance was evaluated using a four-ball tribotester and a Universal Micro-Tribotester (UMT) reciprocating tester. Moreover, the worn surfaces were analyzed using confocal scanning optical microscopy (CSOM), scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS), and Raman spectroscopy. The results showed that refortified oils provided good physical and chemical properties, close to those of fresh oils, and produced better tribological characteristics than used and even fresh oils. There were compositional differences between the films of fresh, used, and refortified oils.

Dense and porous titanium substrates with a biomimetic calcium phosphate coating

The present work studied a biomimetic method using a simplified solution (SS) with calcium and phosphorus ions for coating titanium substrates, in order to improve their bioactivity. Commercially pure titanium dense sheet, microporous and macroporous titanium samples, both produced by powder metallurgy, were treated in NaOH solution followed by heat-treating and immersed in SS for 7, 14 or 21 days. The samples characterization was performed by quantitative metallographic analysis, confocal scanning optical microscopy, scanning electron microscopy, energy dispersive spectroscopy and low angle X-ray diffraction. The results showed coatings with calcium phosphate precipitation in all samples, with globular or plate-like morphology, typical of hydroxyapatite and octacalcium phosphate, respectively, indicating that the solution (SS) has potential for coating titanium substrates. In addition, the different surfaces of substrates had an effect on the formed calcium phosphate phase and thickness of coatings, depending on the substrate type and imersion time in the simplified solution.

Optical equivalence between phase-diversity homodyne detection microscopy and confocal microscopy

We investigated the optical properties of a homodyne scanning optical microscope (HSOM) with phase-diversity detection. We found that an HSOM is a coherent imaging system and has the same coherent transfer function as a confocal scanning optical microscope (CSOM). In addition, the depth discrimination ability of an HSOM is the same as that of a CSOM. The resolution of an HSOM can be improved by inserting an annular aperture in the optical path of either the signal or reference beams without reducing the signal intensity.