Abstract Scanning electron microscopy (SEM) of fibrin fibers typically requires time-consuming preparation and conductive sputter coating, which can alter nanoscale structural measurements. Additionally, protocols for purified fibrinogen clots often include unnecessary steps adapted from plasma clot methods. We aimed to evaluate indium tin oxide (ITO) as a flat, conductive SEM substrate, investigate sputter coating effects on fiber diameter, and simplify purified fibrinogen clot preparation. Platelet-poor plasma and purified fibrinogen clots were formed on ITO substrates and imaged via SEM after 0, 45, or 90 s of sputter coating to quantify diameter variations. For purified clots, a simplified protocol—forming and imaging clots directly on ITO—was compared against a standardized workflow involving clot transfer from microtube lids to carbon tape. Sputter coating duration significantly affected fiber measurements, with longer coating times increasing apparent diameters. Plasma and purified clots showed distinct responses. Crucially, the simplified ITO protocol produced fiber diameter measurements that did not differ significantly from the standardized workflow under identical coating conditions. ITO is an effective conductive substrate that simplifies purified fibrinogen clot preparation by eliminating washing and transfer steps. It yields comparable diameter measurements to standard methods, offering an efficient platform for analyzing fibrin structures.
Cartilage regeneration remains a significant challenge due to the tissue's avascular nature and limited chondrocyte regenerative capacity. While mesenchymal stromal cells (MSCs) offer therapeutic promise, their specific role in cartilage remains undefined. This study characterized mesenchymal-like cells in quail embryo cartilage using histochemical staining, immunohistochemistry, and transmission electron microscopy (TEM). A unique population of cartilage-resident progenitor-like cells with flattened, migratory morphologies was identified. These cells co-expressed stemness markers (CD117, CD34), vascularization (VEGF), remodeling (MMP-9), phagocytosis (CD68), and the chondrogenic marker type II collagen. TEM revealed abundant rough endoplasmic reticulum and lysosomes, confirming active metabolic states. Quantitative analysis showed significant differences in marker expression, with CD34 showing the highest expression (p < 0.05). While these findings demonstrate a multifunctional profile, the study remains primarily descriptive and observational. It provides a foundation for future functional investigations and suggests a previously underappreciated role for this progenitor population, offering a mechanistic basis for advancing MSC-based cartilage regeneration therapies.
Abstract Mapping of ceria oxidation states using electron energy loss spectroscopy has previously been limited to electron flux above the critical threshold for reduction of ceria surfaces (∼3 × 105 e−nm−2s−1). Here, direct electron detection was applied to map the oxidation state of cerium in ceria nanoparticles at low flux (1.25 × 105 e−nm−2s−1). When utilizing direct electron detection, sufficient signal-to-noise was obtained to quantify the native cerium oxidation state in nanometer-resolution maps, which was found to be predominantly Ce4+, but varies significantly based on the sample preparation method. Additionally, local surface reduction of ceria by supported palladium nanoparticles was mapped and quantified. These findings provide insights into the influence of preparation method and palladium incorporation on ceria reducibility and surface chemistry.
Abstract Scanning transmission electron microscopy (STEM) provides atomic-column images for structural and compositional analysis, but resolution is limited by probe broadening, residual aberrations, scan instability, specimen-dependent scattering, and noise. Recent computational approaches for recovering high-spatial-frequency information from experimental images include electron ptychography, machine-learning-based super-resolution, and deconvolution. Deconvolution provides a practical and physically interpretable route for conventional annular dark-field STEM because it directly uses an image-formation model and does not require additional four-dimensional data acquisition or training datasets. However, its reliability is governed by the point spread function used for reconstruction. We present an effective point spread function estimation workflow using an atomically thin two-dimensional reference. A monolayer MoS2 reference was used to reduce thickness-related multiple scattering and specimen-specific broadening, and its preprocessed experimental image was compared with a zero-aberration simulated image through gradient-based optimization of an effective point spread function. The estimated effective point spread function was applied to Si [110] and Nd2Fe14B [100] images acquired under closely matched imaging conditions, extending detectable Fourier information in Si and revealing an intensity feature at the expected Fe-column position between Nd columns. These results demonstrate that the proposed approach can serve as a complementary method for improving the interpretability of conventional STEM imaging.
Abstract Preventing shrinkage of biological tissues during X-ray microfocus computed tomography (3D-microCT) is essential to prevent motion artifacts and microstructural changes, which is particularly problematic in the context of 4D-microCT. While liquid immersion and parafilm wrapping are commonly used to limit dehydration, both methods present limitations for combining them with in situ mechanical testing. This work introduces an in-house environmental stage capable of controlling temperature and relative humidity (RH) around the sample during imaging with vapor phase method. Using 3D-microCT with stained porcine aortic tissues, we assessed the impact of temperature and RH on tissue volume change over time. Results demonstrated that both temperature and RH significantly influence shrinkage rate, with temperature playing a dominant role. Volume loss increased with decreasing RH and increasing temperature. Furthermore, shrinkage was heterogeneous and was particularly pronounced at cut tissue edges. Among all tested conditions, 10°C combined with 95% RH best preserved tissue hydration, showing minimal shrinkage and reduced motion artifacts. Validation at higher spatial resolution confirmed that this controlled environment enables prolonged acquisitions while maintaining image quality superior to imaging under ambient conditions. This study highlights the importance of environmental control during microCT imaging of soft tissues and provides a quantitative framework for selecting optimal atmospheric conditions.
