Application Of Microscopy Research Articles

Amphiphilic coumarin-based probes for live-cell STED nanoscopy of plasma membrane

Plasma membranes are vital biological structures, serving as protective barriers and participating in various cellular processes. In the field of super-resolution optical microscopy, stimulated emission depletion (STED) nanoscopy has emerged as a powerful method for investigating plasma membrane-related phenomena. However, many applications of STED microscopy are critically restricted by the limited availability of suitable fluorescent probes. This paper reports on the development of two amphiphilic membrane probes, SHE-2H and SHE-2N, specially designed for STED nanoscopy. SHE-2N, in particular, demonstrates quick and stable plasma membrane labelling with negligible intracellular redistribution. Both probes exhibit outstanding photostability and resolution improvement in STED nanoscopy, and are also suited for two-photon excitation microscopy. Furthermore, microscopy experiments and cytotoxicity tests revealed no noticeable cytotoxicity of probe SHE-2N at concentration used for fluorescence imaging. Spectral analysis and fluorescence lifetime measurements conducted on probe SHE-2N using giant unilamellar vesicles, revealed that emission spectra and fluorescence lifetimes exhibited minimal sensitivity to lipid composition variations. These novel probes significantly augment the arsenal of tools available for high-resolution plasma membrane research, enabling a more profound exploration of cellular processes and dynamics.

Microstructural Analysis of Stabilized Rammed Earth Using XRD And SEM Methods

The microstructure analysis of stabilized rammed earth (SRE) is crucial for understanding its mechanical properties, durability, and long-term performance. This literature review paper investigates the application of X-ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) in analyzing the microstructural characteristics of SRE. XRD is employed to identify the crystalline phases present in the material, providing insights into the mineralogical composition and the effects of stabilization agents. SEM offers high-resolution images that reveal the surface morphology, particle distribution, and bonding mechanisms within the stabilized matrix. By synthesizing recent studies, this review highlights the advancements in microstructural analysis techniques and their implications for the optimization of SRE in construction. The findings from XRD and SEM analyses are integrated to present a comprehensive understanding of the microstructure-property relationships in SRE. The literature suggests that stabilization methods, such as the addition of cement, lime, or other additives, significantly influence the crystallinity, particle interlocking, and pore structure of the rammed earth. This review discusses the correlation between microstructural features observed through XRD and SEM and the resulting mechanical properties, such as compressive strength and durability. The paper concludes by identifying gaps in the current research and proposing directions for future studies, emphasizing the need for standardized testing protocols and the exploration of novel stabilization materials to enhance the performance of SRE. Keywords: X-Ray Diffraction, Scanning Electron Microscopy, Energy Dispersive X-Ray Analysis

11.7T Diffusion Magnetic Resonance Imaging and Tractography to Probe Human Brain Organoid Microstructure

BackgroundHuman brain organoids are 3-dimensional cellular models that mimic architectural features of a developing brain. Generated from human induced pluripotent stem cells, these organoids offer an unparalleled physiologically relevant in vitro system for disease modeling and drug screening. In the current study, we sought to establish a foundation for a magnetic resonance imaging (MRI)–based, label-free imaging system that offers high-resolution capabilities for deep tissue imaging of whole organoids. MethodsAn 11.7T Bruker/89 mm microimaging system was used to collect high-resolution multishell 3-dimensional diffusion images of 2 induced pluripotent stem cell–derived human hippocampal brain organoids. The MRI features identified in the study were interpreted on the basis of similarities with immunofluorescence microscopy. ResultsMRI microscopy at ≤40 μm isotropic resolution provided a 3-dimensional view of organoid microstructure. T2-weighted contrast showed a rosette-like internal structure and a protruding spherical structure that correlated with immunofluorescence staining for the choroid plexus. Diffusion tractography methods can be used to model tissue microstructural features and possibly map neuronal organization. This approach complements traditional immunohistochemistry imaging methods without the need for tissue clearing. ConclusionsThis proof-of-concept study shows, for the first time, the application of high-resolution diffusion MRI microscopy to image 2-mm diameter spherical human brain organoids. Application of ultrahigh-field MRI and diffusion tractography is a powerful modality for whole organoid imaging and has the potential to make a significant impact for probing microstructural changes in brain organoids used to model psychiatric disorders, neurodegenerative diseases, and viral infections of the human brain, as well as for assessing neurotoxicity in drug screening.

Application of confocal laser microscopy for identification of modern and fossil pollen grains, an example in palm Mauritiinae

Confocal scanning laser microscopy (CSLM) is becoming a powerful tool for palynological studies. CSLM allows palynomorph image sectioning, internal and surface structures visualization, and 3D reconstruction at a higher resolution than standard light microscopy without extra processing. CSLM images are suitable for several image analysis techniques that could help improve the accuracy and reproducibility of taxa identification. Here, using the palm subtribe Mauritiinae (Arecaceae: Calamoideae: Lepidocaryeae) as a model group, we identify modern and fossil pollen grains using CSLM images coupled with ImageJ/Fiji 1.54f plugins and machine learning statistical analyses. Modern taxa pollen grains including Lepidocaryum tenue Mart., Mauritia flexuosa L.f., Mauritiella armata (Mart.) Burret and Mauritiella aculeata (Kunth) Burret were obtained from Smithsonian Tropical Research Institute (STRI) pollen collection or herbarium exsiccates. Fossil pollen of Grimsdalea magnaclavata Germeraad et al. 1968, and Mauritiidites franciscoi (van der Hammen) van der Hammen & Garcia de Mutis 1966, both from Miocene, and Mauritia pollen type from Holocene were obtained from STRI collection. We measured nine shape and exine quantitative parameters, and one qualitative parameter (pollen aperture). Pollen volume was the most important variable (28.270 mean decrease accuracy), followed by pollen aperture (15.003), Skewness (13.466), and spine density (10.246). The machine learning analysis, which included CART and Random Forests, correctly identified both fossil and extant grains. CSLM and the quantitative analysis of morphological traits are a new frontier in palynological studies.

Process development and validation of next generation 3D calibration standards for application in optical microscopy

Abstract A scalable wafer-based fabrication process for a new generation of 3D standards enabling the 3D calibration of optical microscopes is presented and validated. The 3D standards are based on step pyramids with several layers in the µm range and a system of cylindrical knops distributed across the layers as marks for coordinate based calibration. This enables calibration for the three coordinate axes and the orthogonality error between them in a single measurement step. The requirements necessary for such a calibration, as optical non-transparency, reproducible flatness of the pyramid step heights and the lowest possible deviations of the lateral marks coordinates, are met by optimizing the manufacturing process: The deviation of the height steps distributed over the wafer is ±3.6 nm and is primarily caused by the layer deposition processes. The lateral manufacturing accuracy was determined using calibrated scanning electron microscope (SEM) and show a mean deviation of 20 or 60 nm, depending on the lateral size of the structures. The electron beam lithography process and the level of inaccuracy of the SEM standard have an influence on the lateral scaling accuracy. Based on the tactilely generated height values and the coordinates of the mark determined by a calibrated SEM, an example calibration of a confocal laser scanning microscope was successfully performed and showed good conformity to conventional calibration techniques.

Suppression of Subpixel Jitter in Resonant Scanning Systems With Phase-locked Sampling.

Resonant scanning is critical to high speed and in vivo imaging in many applications of laser scanning microscopy. However, resonant scanning suffers from well-known image artifacts due to scanner jitter, limiting adoption of high-speed imaging technologies. Here, we introduce a real-time, inexpensive and all electrical method to suppress jitter more than an order of magnitude below the diffraction limit that can be applied to most existing microscope systems with no software changes. By phase-locking imaging to the resonant scanner period, we demonstrate an 86% reduction in pixel jitter, a 15% improvement in point spread function with resonant scanning and show that this approach enables two widely used models of resonant scanners to achieve comparable accuracy to galvanometer scanners running two orders of magnitude slower. Finally, we demonstrate the versatility of this method by retrofitting a commercial two photon microscope and show that this approach enables significant quantitative and qualitative improvements in biological imaging.

Super-resolution of biomedical volumes with 2D supervision.

Volumetric biomedical microscopy has the potential to increase the diagnostic information extracted from clinical tissue specimens and improve the diagnostic accuracy of both human pathologists and computational pathology models. Unfortunately, barriers to integrating 3-dimensional (3D) volumetric microscopy into clinical medicine include long imaging times, poor depth/z-axis resolution, and an insufficient amount of high-quality volumetric data. Leveraging the abundance of high-resolution 2D microscopy data, we introduce masked slice diffusion for super-resolution (MSDSR), which exploits the inherent equivalence in the data-generating distribution across all spatial dimensions of biological specimens. This intrinsic characteristic allows for super-resolution models trained on high-resolution images from one plane (e.g., XY) to effectively generalize to others (XZ, YZ), overcoming the traditional dependency on orientation. We focus on the application of MSDSR to stimulated Raman histology (SRH), an optical imaging modality for biological specimen analysis and intraoperative diagnosis, characterized by its rapid acquisition of high-resolution 2D images but slow and costly optical z-sectioning. To evaluate MSDSR's efficacy, we introduce a new performance metric, SliceFID, and demonstrate MSDSR's superior performance over baseline models through extensive evaluations. Our findings reveal that MSDSR not only significantly enhances the quality and resolution of 3D volumetric data, but also addresses major obstacles hindering the broader application of 3D volumetric microscopy in clinical diagnostics and biomedical research.

DRY SLIDING WEAR BEHAVIOUR OF CARBON NANOTUBE/ALUMINA/EPOXY HYBRID NANOCOMPOSITES

In the field of materials science, polymer composites have been extensively used in various industries such as marine, automotive, aerospace, sports and other industries due to their good dimensional stability and excellent structural properties. In this present research investigation, epoxy served as the polymer material, while multi-walled carbon nanotubes (MWCNTs) and alumina nanofillers were employed for reinforcing the matrix through hybridization. The wear characteristics of the composite material were examined under dry sliding conditions, employing a pin-on-disc machine with a track diameter of 50 mm. The load on the specimen was varied between low (20 N), medium (40 N) and high (60 N), while the weight fraction of the hybrid nanofillers underwent variations in a range of 0.1–0.5 w/% with an increment of 0.1 w/%. The results showed that the reinforcement of hybrid nanofillers significantly reduces the wear phenomena of the composite material. Hybrid nanocomposites with (0.1, 0.2 and 0.3) w/% of MWCNTs-Al2O3 exhibit noteworthy advancements in the wear resistance. Particularly the 0.3 w/% MWCNTs-Al2O3 hybrid nanocomposite demonstrates exceptional wear resistance compared to pure epoxy. The incorporation of 0.3 w/% of MWCNTs-Al2O3 results in a significantly improved wear resistance, with enhancements of (83, 81 and 80) % observed during low (20 N), moderate (40 N) and high (60 N) loading conditions, respectively, compared to pure epoxy. Similarly, deformation, delamination and filler plugging were observed with medium and high load. The surface morphology of the worn specimens was assessed through the application of field emission scanning electron microscopy.

Self-referencing for quasi shot-noise-limited widefield transient microscopy

Many applications of ultrafast and nonlinear optical microscopy require the measurement of small differential signals over large fields-of-view. Widefield configurations drastically reduce the acquisition time; however, they suffer from the low frame rates of two-dimensional detectors, which limit the modulation frequency, making the measurement sensitive to excess laser noise. Here we introduce a self-referenced detection configuration for widefield differential imaging. Employing regions of the field of view with no differential signal as references, we cancel probe fluctuations and increase the signal-to-noise ratio by an order of magnitude reaching noise levels only a few percent above the shot noise limit. We anticipate broad applicability of our method to transient absorption, stimulated Raman scattering and photothermal-infrared microscopies.

Investigation of irregular apertures and applications in confocal microscopy and speckle imaging

Investigation of irregular apertures and applications in confocal microscopy and speckle imaging

The Pivotal Role of Microscopy in Unravelling the Nature of Microbial Deterioration of Waterlogged Wood: A Review

This review focuses on the pivotal role microscopy has played in diagnosing the type(s) of microbial attacks present in waterlogged ancient wooden objects, and to understand the nature and extent of deterioration of such objects. The microscopic journey began with the application of light microscopy (LM) to examine the deterioration of waterlogged woods, notably foundation piles supporting historic buildings, progressing into the use of high-resolution imaging tools (SEM and TEM) and techniques. Although bacteria were implicated in the deterioration of foundation piles, confirmation that bacteria can indeed degrade wood in its native state came when decaying wood from natural environments was examined using electron microscopy, particularly TEM, which enabled bacterial association with cell wall regions undergoing degradation to be clearly resolved. The information base has been a catalyst, stimulating numerous studies in the past three decades or so to understand the nature of microbial degradation of waterlogged archaeological wood more precisely, combining LM, SEM, and TEM with high-resolution chemical analytical methods, including chemical microscopy. The emerging information is aiding targeted developments towards a more effective conservation of ancient wooden objects as they begin to be uncovered from burial and waterlogging environments.

Nanoscale Phase and Orientation Mapping in Multiphase Polycrystalline Hafnium Zirconium Oxide Thin Films Using 4D-STEM and Automated Diffraction Indexing.

Ferroelectric hafnium zirconium oxide (HZO) holds promise for nextgeneration memory and transistors due to its superior scalability and seamless integration with complementary metal-oxide-semiconductor processing. A major challenge in developing this emerging ferroelectric material is the metastable nature of the non-centrosymmetric polar phase responsible for ferroelectricity, resulting in a coexistence of both polar and non-polar phases with uneven grain sizes and random orientations. Due to the structural similarity between the multiple phases and the nanoscale dimensions of the thin film devices, accurate measurement of phase-specific information remains challenging. Here, the application of 4D scanning transmission electron microscopy is demonstrated with automated electron diffraction pattern indexing to analyze multiphase polycrystalline HZO thin films, enabling the characterization of crystallographic phase and orientation across large working areas on the order of hundreds of nanometers. This approach offers a powerful characterization framework to produce a quantitative and statistically robust analysis of the intricate structure of HZO films by uncovering phase composition, polarization axis alignment, and unique phase distribution within the HZO film. This study introduces a novel approach for analyzing ferroelectric HZO, facilitating reliable characterization of process-structure-property relationships imperative to accelerating the growth optimization, performance, and successful implementation of ferroelectric HZO in devices.

The AMSlide for noninvasive time-lapse imaging of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) symbiosis, the nutritional partnership between AM fungi and most plant species, is globally ubiquitous and of great ecological and agricultural importance. Studying the processes of AM symbiosis is confounded by its highly spatiotemporally dynamic nature. While microscopy methods exist to probe the spatial side of this plant-fungal interaction, the temporal side remains more challenging, as reliable deep-tissue time-lapse imaging requires both symbiotic partners to remain undisturbed over prolonged time periods. Here, we introduce the AMSlide: a noninvasive, high-resolution, live-imaging system optimised for AM symbiosis research. We demonstrate the AMSlide's applications in confocal microscopy of mycorrhizal roots, from whole colonisation zones to subcellular structures, over timeframes from minutes to weeks. The AMSlide's versatility for different microscope set-ups, imaging techniques, and plant and fungal species is also outlined. It is hoped that the AMSlide will be applied in future research to fill in the temporal blanks in our understanding of AM symbiosis, as well as broader root and rhizosphere processes.

Far-field superresolution of thermal sources by double homodyne or double array homodyne detection

We propose two schemes for estimating the separation of two thermal sources via double homodyne and double array homodyne detection considering the joint measurement of conjugate quadratures of the image plane field.By using the Cramér–Rao bound, we demonstrate that the two schemes can estimate the separation well below the Rayleigh limit and have an advantage over direct imaging when the average photon number per source exceeds five.For arbitrary source strengths, double homodyne detection is superior to homodyne detection when the separation is above 25/4σ/N s , σ is the beam width, N s is the average photon number per source.A larger separation can be estimated better via double array homodyne detection with the superiority of flexible operation compared with other schemes. High-speed and high-efficiency detection enables the measurement schemes with potential practical applications in fluorescence microscopy, astronomy and quantum imaging.

Fourier space aberration correction for high resolution refractive index imaging using incoherent light

An aberration correction method is introduced for 3D phase deconvolution microscopy. Our technique capitalizes on multiple illumination patterns to iteratively extract Fourier space aberrations, utilizing the overlapping information inherent in these patterns. By refining the point spread function based on the retrieved aberration data, we significantly improve the precision of refractive index deconvolution. We validate the effectiveness of our method on both synthetic and biological three-dimensional samples, achieving notable enhancements in resolution and measurement accuracy. The method's reliability in aberration retrieval is further confirmed through controlled experiments with intentionally induced spherical aberrations, underscoring its potential for wide-ranging applications in microscopy and biomedicine.

Abstract 114: Inhibition Of Lecithin Cholesterol Acyl Transferase By A Novel Anti-apolipoprotein A1 Antibody Fragment

Apolipoprotein A1 (APOA1), the most abundant protein in high-density lipoproteins (HDL), is the primary activator of lecithin cholesterol acyl transferase (LCAT). LCAT’s cholesteryl esterification activity is key for maturing nascent HDL discs into the spherical forms commonly found in plasma. However, the molecular mechanism for APOA1 activation of LCAT is poorly understood. Using phage display selection against nanodiscs (lipid complexes stabilized by an APOA1 variant commonly used in transmembrane protein structural biology), we have identified, isolated, and expressed a novel Fab fragment (Fab16). Size exclusion chromatography elution shift experiments showed that Fab16 readily bound reconstituted HDL (rHDL) particles containing human purified plasma APOA1 with nearly quantitative binding at a 1:1 molar ratio of Fab16 to APOA1. Negative stain electron microscopy (NSEM), confirmed that 1-2 Fab16 bound to each rHDL particle in a specific pattern around the disc periphery. Additionally, Fab16 effectively bound both large and small spherical HDL particles isolated from human plasma. Chemical cross-linking / mass spectrometry experiments indicated that Fab16 binds an APOA1 epitope between residues 94 and 118 (i.e., within amphipathic helix 4). As our previous work has shown that APOA1 helix 4 is important for LCAT activation, we hypothesized that Fab16 would inhibit LCAT activity. Using a novel gas chromatography cholesterol esterification assay, we found that Fab16 applied at a 1.5:1 molar ratio to APOA1 inhibited LCAT cholesterol esterification activity by 56±7%. The symmetrical nature of both discoidal and spherical HDL particles has hampered the application of cryogenic electron microscopy (cryo EM) for understanding HDL structure. Fab16 may provide a valuable ‘landmark’ to assist in deriving a high-resolution structure of the complex. Work to optimize conditions for collecting high quality cryo EM datasets of discoidal and spherical HDL in the presence and absence of LCAT is ongoing. Deriving a high-resolution understanding of HDL structure and its interaction with remodeling factors such LCAT has been a long-standing problem but is crucial for deriving new strategies to manipulate HDL function for therapeutic benefit.

A Simple Doublet Lens Design for Mid-Infrared Imaging.

Wide-field mid-infrared (MIR) hyperspectral imaging offers a promising approach for studying heterogeneous chemical systems due to its ability to independently characterize the molecular properties of different regions of a sample. However, applications of wide-field MIR microscopy are limited to spatial resolutions no better than ∼1 μm. While methods exist to overcome the classical diffraction limit of ∼λ/2, chromatic aberration from transmissive imaging reduces the achievable resolution. Here we describe the design and implementation of a simple MIR achromatic lens combination that we believe will aid in the development of resolution-enhanced wide-field MIR hyperspectral optical and chemical absorption imaging. We also examine the use of this doublet lens to image through polystyrene microspheres, an emerging and simple means for enhancing spatial resolution.

Application of dermoscopy and reflectance confocal microscopy in vivo in the evaluation of nevi in children.

Melanocytic nevi are frequently observed in the pediatric population. While newly acquired nevi can appear during childhood, congenital nevi can continue to grow and clinically change, making patient caregivers concerned. Reflectance confocal microscopy (RCM) in vivo is a noninvasive tool that might enhance the diagnostic accuracy of dermoscopy, reducing the rate of unnecessary surgical procedures. This study aimed to assess the utility of RCM in increasing the diagnostic accuracy of pediatric melanocytic nevi that show pigmentation changes or grow rapidly. Pediatric patients who presented between January 2022 and February 2023 in a single institution with rapidly growing melanocytic nevi or nevi that presented changes in the pigmentation were included in the study. All nevi were evaluated by means of dermoscopy and RCM. Forty-two patients with a total of 42 melanocytic nevi were included. Most lesions showed a honeycombed pattern (n = 21, 50%). On RCM, only 3 of 42 nevi presented atypical cells within the epidermis (7.1%). Evaluation of the dermoepidermal junction (DEJ) revealed the predominance of the meshwork pattern (n = 22, 52.4%). Notably, features considered significant for atypical melanocytic nevi included 9 nevi with scant atypical melanocytes (21.4%) and 3 nevi with nonedge papillae (7.1%). None of the studied lesions required biopsy among this cohort. Most rapidly growing and clinically changing nevi rarely exhibit single atypical cells in the DEJ. The RCM served as a valuable adjunct to dermoscopy, allowing reassurance in the evaluation of these lesions.

Probing molecular architectures and interactions with scanning tunneling microscopy on graphite and arachidic acid functionalized surfaces

This study investigates the application of scanning tunneling microscopy in exploring molecular structures and interfaces relevant to nanotechnology. Graphite was selected as a sample due to its ease of visualization of atomic arrangements and surface inertness. The surface of highly oriented pyrolytic graphite treated with arachidic acid (C20H32O2) was examined to gain insights into the behavior of organic molecules at liquid-solid interfaces. Through detailed observations, the study demonstrates the versatility of scanning tunneling microscopy in elucidating molecular architectures and interactions. The findings underscore the importance of scanning tunneling microscopy in advancing our understanding of molecular systems and driving progress in nanotechnology applications. This work highlights the pivotal role of scanning tunneling microscopy in unraveling the complexities of nanoscale phenomena and fostering future innovations in the field.

Structural Characterization of La0.6Sr0.4CoO3-δ Thin Films Grown on (100)-, (110)-, and (111)-Oriented La0.95Sr0.05Ga0.95Mg0.05O3-δ.

In this study, a detailed structural characterization of epitaxial La0.6Sr0.4CoO3-δ (LSC) films grown in (100), (110), and (111) orientations was conducted. LSC is a model air electrode material in solid oxide fuel and electrolysis cells and understanding the correlation of bulk structure and catalytic activity is essential for the design of future electrode materials. Thin films were grown on single crystals of the perovskite material La0.95Sr0.05Ga0.95Mg0.05O3-δ cut in three different directions. This enabled an examination of structural details at the atomic scale for a realistic material combination in solid oxide cells. The investigation involved the application of atomic force microscopy, X-ray diffraction, and high-resolution transmission electron microscopy to explore the distinct properties of these thin films. Interestingly, ordering phenomena in both cationic as well as anionic sublattices were found, despite the fact that the thin films were never at higher temperatures than 600 °C. Cationic ordering was found in spherical precipitates, whereas the ordering of oxygen vacancies led to the partial transition to brownmillerite in all three orientations. Our results indicate a very high oxygen vacancy concentration in all three thin films. Lattice strains in-plane and out-of-plane was measured, and its implications for the structural modifications are discussed.