Microscopic Samples Research Articles

Basic properties of solidified organic liquids at a cryogenic temperature for electron microscopic visualization and sample preparation of dispersion systems.

It is challenging to image structures in liquids for electron microscopy (EM); thus, low-temperature imaging has been developed, initially for aqueous systems. Organic liquids (OLs) are widely used as dispersants, although their cryogenic EM (cryo-EM) imaging is less common than that of aqueous systems. This is because the basic properties (e.g., vapor pressure, density, and amorphousness) of OL in the solid state have not been extensively investigated, preventing the determination of whether or not the observed structure is free from artifacts. Herein, I summarized physical data related to the phase change, and the solid density at 77 K and sublimation speed for some OLs were measured independently, to discuss the applicability of OLs for cryo-EM. Among various OL properties, the sublimation temperature, pressure, and rate, and crystallinity are important for cryo-EM. The sublimation-related properties are used to judge whether the OL is stable during storage, observation, and sample preparation such as etching. These properties were calculated, and the calculated sublimation speed matched with that measured by cryo-SEM movie imaging. Crystallinity was estimated using the difference between the extrapolated temperature-dependent liquid density and the solid density of frozen OLs measured in liquid nitrogen. Artifacts observed upon freezing were exemplified by focused ion beam cross-sections of OL-in-water emulsions, and cracks, voids, and wrinkles are found in the OL phase at a large shrinkage ratio. The study findings show that the applicability of OLs largely differs for structural isomers and that appropriate OLs are required for the cryo-EM imaging of nonaqueous systems.

Image-guided optogenetic spatiotemporal tissue patterning using μPatternScope

In the field of tissue engineering, achieving precise spatiotemporal control over engineered cells is critical for sculpting functional 2D cell cultures into intricate morphological shapes. In this study, we engineer light-responsive mammalian cells and target them with dynamic light patterns to realize 2D cell culture patterning control. To achieve this, we developed μPatternScope (μPS), a modular framework for software-controlled projection of high-resolution light patterns onto microscope samples. μPS comprises hardware and software suite governing pattern projection and microscope maneuvers. Together with a 2D culture of the engineered cells, we utilize μPS for controlled spatiotemporal induction of apoptosis to generate desired 2D shapes. Furthermore, we introduce interactive closed-loop patterning, enabling a dynamic feedback mechanism between the measured cell culture patterns and the light illumination profiles to achieve the desired target patterning trends. Our work offers innovative tools for advanced tissue engineering applications through seamless fusion of optogenetics, optical engineering, and cybernetics.

Fluorescence microscopic image enhancement method based on multi-saliency guided filtering fusion

Abstract Aiming at addressing the issues of colour deviation and clarity reduction in digital pathological imaging systems caused by complex environmental interferences such as uneven slice thickness, sample jitter, and background noise, this paper proposes a multi-saliency-guided filtering fusion enhancement method that combines energy saliency and Gaussian saliency. Firstly, a series of Gamma correction and spatial linear saturation adjustments are applied to enhance the contrast and colour saturation of blurred images, resulting in a sequence of pseudo-exposure images with improved contrast and saturation. Then, the pseudo-exposure images are decomposed into base and detail layers, and the fusion rules of energy saliency and Gaussian saliency-guided filtering are applied to each layer separately. Finally, clear microscopic images are obtained through two-scale reconstruction. This method overcomes the limitations of conventional deblurring algorithms that rely on prior estimation of scene depth and complex depth mapping processes. Experimental results on a dataset of cytological bright-field microscopic imaging samples show that the clarity evaluation metrics, including peak signal-to-noise ratio, root mean square error, and structural similarity index, have significantly improved, indicating that the proposed method can effectively improve the colour fidelity of the samples while eliminating noise interference and enhancing the clarity of cellular texture structures.

Image Analysis in Histopathology and Cytopathology: From Early Days to Current Perspectives.

Both pathology and cytopathology still rely on recognizing microscopical morphologic features, and image analysis plays a crucial role, enabling the identification, categorization, and characterization of different tissue types, cell populations, and disease states within microscopic images. Historically, manual methods have been the primary approach, relying on expert knowledge and experience of pathologists to interpret microscopic tissue samples. Early image analysis methods were often constrained by computational power and the complexity of biological samples. The advent of computers and digital imaging technologies challenged the exclusivity of human eye vision and brain computational skills, transforming the diagnostic process in these fields. The increasing digitization of pathological images has led to the application of more objective and efficient computer-aided analysis techniques. Significant advancements were brought about by the integration of digital pathology, machine learning, and advanced imaging technologies. The continuous progress in machine learning and the increasing availability of digital pathology data offer exciting opportunities for the future. Furthermore, artificial intelligence has revolutionized this field, enabling predictive models that assist in diagnostic decision making. The future of pathology and cytopathology is predicted to be marked by advancements in computer-aided image analysis. The future of image analysis is promising, and the increasing availability of digital pathology data will invariably lead to enhanced diagnostic accuracy and improved prognostic predictions that shape personalized treatment strategies, ultimately leading to better patient outcomes.

Orientation-dependent deformation and failure of micropillar shape memory ceramics: A 3D phase-field study

Some microscopic samples of zirconia-based shape memory ceramics (SMCs) have shown full martensitic phase transformation (MPT) over multiple loading cycles without cracking. However, the occurrence of MPT is strongly influenced by grain orientation. Depending on the specific grain orientation relative to the loading direction, alternative mechanisms such as plastic slip and fracture may emerge. This study introduces a phase-field (PF) based framework that integrates a PF-MPT model, a PF fracture model, and a crystal viscoplasticity model to investigate the effects of grain orientation on MPT, plastic slip, and fracture mechanisms in SMC micropillars. Single crystal micropillars are created to distinguish the orientations that facilitate each mechanism. A wide range of grain orientations are found to predominantly exhibit MPT. Micropillars with grain orientations close to the (100) and (001) directions primarily experience fracture, with minimal plastic slip. Additionally, samples oriented along the (110) direction show a significant amount of plastic slip. A pole figure is constructed to elucidate the interplay between MPT, cracking, and plastic slip under compressive loading conditions. This research provides valuable insights into the intricate behavior of SMCs under different loading scenarios, crucial for optimizing their performance in practical applications.

Coupling nanoscopic tomography and micromagnetic modelling to assess the stability of geomagnetic recorders

The recording of planetary magnetic fields is often attributed to uniformly-magnetised nanoscopic iron oxides, called single-domain. Yet, the main magnetic constituents of rocks are more complex, non-uniformly magnetised grains in single or multi-vortex states. We know little about their behaviour due to limitations in defining their precise shape and internal magnetic structure. Here we combine non-destructive Ptychographic X-ray Computed Nano-tomography with micromagnetic modelling to explore the magnetic stability of remanence-bearing minerals. Applied to a microscopic rock sample, we identified hundreds of nanoscopic grains of magnetite/maghemite with diverse morphologies. Energy barrier calculations were performed for these irregularly shaped grains. For some grains, these morphological irregularities near the transition from single-domain to the single-vortex state allow for multiple domain states, some unstable and unable to record the field for significant periods. Additionally, some other grains exhibit temperature-dependent occupancy probabilities, potentially hampering experiments to recover the intensity of past magnetic fields.

Perception of audified acoustic emissions from compression of microscopic samples

Recording of acoustic emissions (elastic waves emitted by dislocation motion) during compression of microscopic samples is a novel emerging tool for the investigation of mechanical properties of materials. Yet the resulting signals can be so noisy and complex that they don't easily allow for identifying the deformation mode or the material involved. This study aims at testing if the human auditory system can pick patterns in the audified recordings (i.e. transposed from the ultrasonic to the audible range) that match the physical characteristics of the samples. In a Free Sorting Task, participants sorted sounds into groups according to perceived similarity. The sounds originated in recordings varying according to material, deformation mode, and size of the sample. During clustering and multidimensional analysis, sounds were found to be first grouped according to spectral features: Zinc and Magnesium recordings respectively produced high-pitched and low-pitched sounds. Sounds were further discriminated according to perceived sound level (soft vs. loud for slipping vs. twinning) and number of perceived sound events (no clearly associated physical parameter). Correlations were found between subjective groupings and audio descriptors, so that audio-inspired mechanical investigations can now be envisioned.

Enhanced MALDI-2 Sensitivity with Reflecting Post-Ionization Laser for High-Resolution MS Imaging Combined with Real-Time Microscope Imaging.

Laser-induced matrix-assisted laser desorption/ionization post-ionization (MALDI-2) could improve the MALDI sensitivity of biological metabolites by over 1 order of magnitude. Herein, we demonstrate that MALDI-2 sensitivity can be further enhanced with reflecting post-ionization laser that multiplies the intersection times between laser and MALDI plume. This method, which we named MALDI-2+, typically brought over 2 times sensitivity improvement from conventional MALDI-2. Advancing in sensitivity thereby prompted us to pursue higher mass spectrometry imaging (MSI) spatial resolution. A dedicated T-shaped ion guide was designed to allow perpendicular incidence of ablation laser in reflection geometry MALDI. Although 8-10 μm pixel was used in MALDI imaging due to the limited precision of the motorized stage, the laser spot diameter could be down to 2.5 μm for potentially higher spatial resolution. In addition, this ion source enabled real-time and high-quality microscope imaging from backward of the sample plate. Beneficially, we were able to monitor the actual laser spot condition in real time as well as obtain high-resolution microscopic sample images that inherently register with MSI images. All of these benefits have been demonstrated by analyzing standard samples and imaging of cells. We believe that the enhancement in sensitivity, spatial resolution, and microscope capacity of our design could facilitate spatial omics studies.

Influence of Initial Film Properties in UVO-Mediated Patterning of an Elastomeric Film Using a TEM Grid.

Ultraviolet irradiation of a cross-linked polydimethylsiloxane (PDMS) Sylgard 184 film in the presence of atmospheric oxygen (UVO) through a bare transmission electron microscope (TEM) sample holding grid is a rather simple and widely utilized technique for creating micropatterned surfaces. The surface oxidation of a Sylgard 184 film due to UVO exposure is associated with densification and the formation of a silica-like surface layer, which under a TEM grid happens only over the exposed areas of the film, resulting in a physicochemical pattern. It is known that the depth (hD) of the features depends on the duration of UVO exposure (tE). In this article, we show for the first time that hD also depends on the initial film thickness (hF) and the cross-linker percentage (CL, ratio of part A to part B) in a Sylgard 184 thin film. We show that for a specific tE, hD progressively decreases with the reduction in hF. On the other hand, hD shows a nonmonotonic dependence with CL, resulting in patterns with maximum depth for CL ≈ 10.0%. We attribute this observation to the combined effect of resistance against the penetration of the propagation front by the rigid substrate as well as stress relaxation within the exposed parts of the film below the propagating front in films with higher CL values leading to the variation of hD. The observation reported here would allow the potential fabrication of polymer films with physicochemical patterns with feature height on demand by a one-step, facile technique.

Polar magneto-optical Kerr effect spectroscopy with a microscope arrangement for studies on 2D materials.

We describe a setup for magneto-optical Kerr effect (MOKE) spectroscopy suitable for Kerr rotation (ϕ) and ellipticity (η) measurement on microscopic samples, such as flakes of two-dimensional materials. A spatial resolution of ∼25μm, limited by the demagnified monochromator exit slit image, was achieved. The use of mirrors allows for measurement in polar MOKE geometry with a conventional electro-magnet, without requiring holes in the magnet pole pieces. The microscope-like optics also has a 90° twisted periscope arrangement of two mirrors that helps transport light without change in its circular polarization state. A Jones matrix analysis of the setup brings out the influence of the beam-splitter on the measured signals. Its correction requires the ellipsometry parameters of the beam-splitter in transmission mode, which were measured separately. The working of the setup is tested by measuring the ϕ and η spectra of 2H-WS2 flakes at low temperature, verifying them using Kramers-Kronig analysis and extracting the Landé g-factor of the ground state exciton from them.

Operando Liquid-cell Transmission Electron Microscope Sample Holder with Bulk Reference and Counter Electrodes for Electrocatalysis Applications

Operando Liquid-cell Transmission Electron Microscope Sample Holder with Bulk Reference and Counter Electrodes for Electrocatalysis Applications

Investigation of the parasitic protozoa that cause diarrhea and their effect on GOT, GPT and ALP

Abstract The current training was accompanied throughout the dated from December 2021 until June 2022 to study the spread of parasitic protozoa in children under the age of six and to study the biochemical changes, This current training was accompanied at the College of Science, Al-Qadisiyah University, in cooperation with the Maternity and Children’s Hospital in Kut and the Maternity and Children’s Hospital in Diwaniyah. 350 microscopic stool samples from children aged (0-6) years were examined. The samples that were diagnosed under a microscope and containing the study parasites (50 samples), Venous blood (5ml) from all samples was collected to measure some biochemical parameters. The outcomes of the existing training displayed that the highest contagion with parasites protozoa was in the month of July, and the parasite Entamoeba histolytica recorded the highest infection among the rest of the protozoan parasites. It included 13 out of 85 samples with a ratio of (15.29) positive for parasite infection, while our current study recorded the lowest infection in March, as it included that 5 of 38 (13.15) were positive for intestinal parasites. The study showed that there were changes in the biochemical parameters that were studied paralleled to the results of the controller group, Changes in the three liver enzymes GOT, GPT and ALP were studied. The results of the showed a high concentration of enzymes the three livers GOT, GPT and ALP.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Magnetic flux trapping in porous high-Tc superconductors

Porosity affects the properties of high-Tc superconductors and can improve their performance by enhancing oxygenation, cryocooling, etc. Among other factors, the presence of pores plays a significant role in the process of magnetic flux trapping. Relationships with the porosity manifest in the irreversibility field, the full penetration field, and the remnant magnetization of the samples. To account for the effect of porosity on the trapped magnetic flux into type-II superconductors, a simple toy model is suggested. Generally, as the porosity increases, the trapped flux and related parameters tend to diminish. However, in the case of microscopic samples, porosity can enhance magnetic flux trapping.

Pseudo-marginal approximation to the free energy in a micro-macro Markov chain Monte Carlo method.

We introduce a generalized micro-macro Markov chain Monte Carlo (mM-MCMC) method with pseudo-marginal approximation to the free energy that is able to accelerate sampling of the microscopic Gibbs distributions when there is a time-scale separation between the macroscopic dynamics of a reaction coordinate and the remaining microscopic degrees of freedom. The mM-MCMC method attains this efficiency by iterating four steps: (i) propose a new value of the reaction coordinate, (ii) accept or reject the macroscopic sample, (iii) run a biased simulation that creates a microscopic molecular instance that lies close to the newly sampled macroscopic reaction coordinate value, and (iv) microscopic accept/reject step for the new microscopic sample. In the present paper, we eliminate the main computational bottleneck of earlier versions of this method: the necessity to have an accurate approximation of free energy. We show that the introduction of a pseudo-marginal approximation significantly reduces the computational cost of the microscopic accept/reject step while still providing unbiased samples. We illustrate the method's behavior on several molecular systems with low-dimensional reaction coordinates.

Recent Advances and Current Trends in Transmission Tomographic Diffraction Microscopy.

Optical microscopy techniques are among the most used methods in biomedical sample characterization. In their more advanced realization, optical microscopes demonstrate resolution down to the nanometric scale. These methods rely on the use of fluorescent sample labeling in order to break the diffraction limit. However, fluorescent molecules' phototoxicity or photobleaching is not always compatible with the investigated samples. To overcome this limitation, quantitative phase imaging techniques have been proposed. Among these, holographic imaging has demonstrated its ability to image living microscopic samples without staining. However, for a 3D assessment of samples, tomographic acquisitions are needed. Tomographic Diffraction Microscopy (TDM) combines holographic acquisitions with tomographic reconstructions. Relying on a 3D synthetic aperture process, TDM allows for 3D quantitative measurements of the complex refractive index of the investigated sample. Since its initial proposition by Emil Wolf in 1969, the concept of TDM has found a lot of applications and has become one of the hot topics in biomedical imaging. This review focuses on recent achievements in TDM development. Current trends and perspectives of the technique are also discussed.

Study on scale formation and corrosion behavior of heat exchanger steel 20 at different temperatures

This study investigates the scaling and corrosion behavior of steel 20 of cooling water in two locations: oil depot working area and living quarters. Furthermore, it explores the effect of temperature on scaling rate and corrosion rate. Through static scaling test and dynamic scaling prediction, the scaling effect of water quality is analyzed. The results show that the scaling degree of two water quality conditions is different with the change of temperature. The water scaling initiates at 60 °C in the living quarters, while a slight amount of scaling occurs when the temperature reaches 75 °C at working area. Through the utilization of open circuit potential, polarization curve analysis, scanning electron microscope (SEM) imaging and X-ray diffraction, the corrosion potential, corrosion rate, macroscopic and microscopic corrosion samples, as well as corrosion products of steel 20 were comprehensively examined. It was found that, in the temperature range of 30 −50 °C, the corrosion rate of steel 20 in the working area showed a linear trend with water temperature, while the corrosion rate in the living quarters exhibited an exponential function trend with temperature. Considering the combined impact of scaling and corrosion on heat exchanger performance, if the operating conditions can exceed 40 °C, the water quality within the working area poses minimal risks and operational pressure to the heat exchanger. Therefore, it is more suitable as a cooling water source compared to that in living quarters.

Structure probing by holographic imaging at nanometer scale with X-ray lasers (SPHINX)

The SPHINX project aims generating femtosecond-exposure X-ray holograms of microscopic samples and of their internal parts with nanometer resolution. The application is based on a new implementation of the phase-contrast holography that overcomes the main limitations encountered in the current systems (most based on absorption-contrast), namely the low energy range, the limited detector granularity and the weak illumination. The combination of polycapillary lenses, large X-Ray CCD arrays with small pixel size and XFEL sources allows splitting the beam, focusing, magnification and phase-contrast imaging in the keV energy range. Due to the wavelength-dependent optical behavior of the samples, the refractive diffraction reduces the diffraction limit together with a fast drop of the characteristic angles, both essential for the resolving power (given the limited X-ray detector pitch), while also eliminating the shadow effect and giving access to full structure probing. The key parameters are defined by the focusing optics, which could be, according to the beam and sample sizes, a polycapillary micro semi-lens or a combination of the former with a parabolic monocapillary. The advantage of “non-perfect” optics (not providing a point-like focus) is their divergence, driven mostly by the single waveguide. This allows sending on the same detector area the diverging object and reference beams (Fresnel configuration), both provided by a single optical element, condition unreachable with a Fresnel lens or a crystal mirror. Moreover, the femtosecond exposure time on X-FELs permits holographic reconstruction of in-vivo cell elements, viruses and nanorobotic devices during ultrafast molecular processes, yet unexplored by imaging techniques.

Quantitative Approach for Anemia Detection Using Regression Analysis

Anemia, generally termed as deficiency of hemoglobin or red blood cells in the blood is significant global health concern for the population in underdeveloped as well as in developing nations specially, for children and young women in rural areas. This paper proposes a quantitative approach for anemia detection by regression analysis technique which predicts hemoglobin level in the blood. To achieve this, the image dataset of microscopic blood sample is collected from 70 individuals. The data collection requires proper procedure as it plays vital part in system implementation. The statistical feature utilizing mean pixel intensity values from the red, green, and blue color planes of the images are given as input to the regression model. For the proposed system, we have employed multiple regression analysis model using machine learning approach with both three and four regression coefficients to establish relation between features obtained from blood samples and the hemoglobin level in the blood to achieve the specified task of anemia detection in an individual. Performance analysis show promising results for the proposed system with co-efficient of determination (R2) and root mean square error (RMSE) found out be 0.923 and 1.682 respectively. Overall, this paper presents valuable system for anemia detection based on hemoglobin estimation which can be implemented in areas with limited medical resources and gives another supportive technological solution for current healthcare problems.

Pre- and Postembedding Immunoelectron Microscopy to Analyze the Central Nervous System.

Immunocytochemistry, a method of delineating the subcellular localization of target proteins, was developed from immunohistochemistry. In principle, proteins are labeled using an antigen-antibody reaction. In order to observe under an electron microscope, the reaction product must scatter the electron beam with sufficient contrast while it is necessary to have an amplifying label that can withstand the observation. We have some detailed tips on making electron microscope samples to achieve this objective, and we would be happy to help you.