Microscopy and Microanalysis最新文献

Transmission electron microscopy offers atomic resolution, which is critical for analyzing nanomaterials. However, its two-dimensional nature limits its ability to capture full three-dimensional (3D) morphologies, which has motivated the development of 3D techniques such as electron tomography. While these techniques improve visualization, they are constrained by beam sensitivity, which can damage materials during prolonged exposure. In this study, we demonstrated the efficacy of the Frame Interpolation for Large Motion (FILM) method -augmenting STEM tilt series by synthesizing intermediate projections between measured tilt images-for enhancing the quality of the 3D reconstruction of nanoparticles using scanning transmission electron microscopy tomography. When applied to a nanooctahedron, a hollow nanotube, and a nanoframe, FILM outperformed traditional linear interpolation, yielding superior image quality assessment scores, particularly for complex structures acquired with large tilt intervals. However, when the structure is relatively simple and the angular sampling is sufficiently dense, FILM interpolation may not yield further improvement and can even oversmooth subtle surface features. This method is particularly promising for the visualization of intricate nanostructures, indicating its potential for various applications in electron tomography.

Reperfusion injury is a paradoxical response following ischemic heart disease that exacerbates myocardial damage, resulting in ischemia reperfusion (IR) injury. This study explored the protective effect of carvedilol (CAR) on cardiac tissue in myocardial IR injury. Forty Wistar rats were randomly allocated into control, IR, and CAR + IR groups. Myocardial IR injury was surgically induced for 45 min followed by 120 min of reperfusion. CAR (2 mg/kg/day) was administered orally for 7 days prior to IR induction. Cardiac effects were assessed via physiological measurements, serum biomarkers of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI), immunohistochemical detection of Bcl-2-associated X-protein (Bax) and B cell lymphoma 2 (Bcl-2), light and electron microscopy, histopathological grading, and morphometric and statistical analysis. CAR pretreatment markedly preserved cardiac function in IR-injured rats by significantly reducing QTc and ST-segment elevations and suppressing ventricular arrhythmias. Biochemically, serum CK-MB and cTnI levels were significantly lowered, while Bax expression decreased and Bcl-2 increased. Histologically, CAR mitigated necrosis, hemorrhage, and cellular degeneration. Ultrastructurally, myocardial integrity, capillary patency, and telocyte morphology were preserved. CAR pretreatment effectively mitigated myocardial IR injury through multifaceted mechanisms including improving cardiac electrical stability, reducing biochemical and apoptotic damage, and preserving histological and ultrastructural integrity.

Finasteride is frequently prescribed for the management of nodular prostatic hyperplasia and androgenic alopecia. However, it has a detrimental impact on spermatogenesis, leading to infertility. Lycopene is a carotenoid with strong antioxidant and antiapoptotic effects. The present investigation evaluated the protective effect of lycopene against finasteride-induced testicular damage in adult rat testes. Four groups were created: control group, lycopene group (4 mg/kg/day), finasteride group (5 mg/kg/day), and lycopene-finasteride group. The agents were administered by oral gavage for 8 weeks. Finasteride significantly reduced the levels of dihydrotestosterone and testosterone. The level of superoxide dismutase markedly decreased, while malondialdehyde increased. The mRNA expression of caspase 3 was significantly elevated, whereas that of BCL2 was markedly diminished. Also the sperm parameters were significantly reduced. Moreover, finasteride induced histopathological changes in the form of irregular, degenerated seminiferous tubules. Vacuolation and desquamation of spermatogenic cells inside the lumen of tubules were evident. A marked rise in the immune expression of Bax was associated with a marked reduction in the expression of BCL2 and PCNA. Interestingly, few changes were detected in rats concomitantly administered lycopene and finasteride. The data suggest that lycopene provides substantial protection against finasteride-induced testicular damage.

Light sheet fluorescent microscopy (LSFM) can be used to obtain large-scale (up to whole organ) 3D images; however, once one turns to lineage tracing, there is a conflict between efficacy of tissue clearing, permeabilization for immunolabeling, and tracing signal salvation. We report a methodological and analytical pipeline that would aid researchers using multi-color (Confetti) lineage tracing and utilize LSFM along with immunolabeling procedures. We used mouse cornea as a primary object and provided its detailed characteristics at a cellular level using dual-view inverted selective plane illumination (diSPIM) microscopy. We developed a protocol for a 3D image acquisition and analysis pipeline of lineage tracing data in Prrx1-expressing cells of corneal stromal and endothelial compartments. Morphometry data included calculation of cell number in epithelium, stroma, and endothelium along with measurement of corneal thickness and radius of curvature. The developed image analysis pipeline allowed calculation of Confetti marker distribution and quantitative clonal expansion estimation with adjustment imposed to correct spectral Confetti channel leakages. We believe that the presented methodology and 3D image analysis scripts should facilitate the lineage tracing studies and may be adapted to other organs.

Accurate characterization of wood microstructure requires high-quality sample preparation, especially for degraded, mineralized, or embedded samples. Through a detailed and systematic investigation, we suggest a simple, fast, and cost-effective method for preparing transverse surfaces of wood for scanning electron microscopy. The methodology used is easily accessible and simple. We compare traditional microtome cutting using both disposable blades and a fixed knife with sanding and polishing techniques. The choice of method is determined by the physical condition and structural characteristics of the sample. Disposable blades, when used with continuous moistening, yielded the best results for reference wood, while the fixed knife proved to be more effective for mineralized or epoxy-embedded samples due to their rigidity and resistance to deflection. Maintaining blade sharpness and surface moisture were critical in all cutting techniques. Polishing proved to be a particularly effective technique for the preparation of degraded, mineral-rich, or heterogeneous samples. In combination with epoxy embedding, it offers a practical alternative to microtome cutting, ensuring the protection of fragile structures, the long-term stability of samples, and the possibility of re-polishing. Epoxy impregnation was easy for degraded wood, while sprucewood required vacuum-pressure treatment. Care must be taken when polishing to avoid reaching non-embedded regions beneath the surface.

Tissue autofluorescence (AF) is a major limitation to immunofluorescence (IF) staining of formalin-fixed paraffin-embedded (FFPE) tissues. This study evaluated the AF reduction efficacy of sodium borohydride (NaBH₄) in FFPE quail (Coturnix coturnix) lung tissues processed under heat-induced epitope retrieval (HIER) or enzyme-induced antigen retrieval (EIAR) conditions. Following antigen retrieval (AR), lung sections were treated with 1 or 2 mg/mL NaBH₄ concentrations and compared with untreated controls. Autofluorescence was evaluated across green and red channels under identical imaging settings; and mean fluorescence intensity (MFI) was quantified using Fiji (ImageJ). Data were analyzed by one-way ANOVA with Tukey's post hoc test. NaBH₄ significantly reduced AF in green and red channels (p < 0.05) under both AR conditions. No difference was observed between 1 and 2 mg/mL treatments. Collagen type-IV were detected in HIER-processed tissues but not in the EIAR-processed ones. These observations shows that NaBH₄ is effective in reducing green and red AF in FFPE quail lungs regardless of AR method employed; and immunostaining of collagen type-IV in FFPE quail lung is better achieved with HIER than with EIAR methods. This study presents an optimized approach for reducing AF and enhancing antigen visualization in IF studies of avian FFPE lung tissues.

This study explores the utility of fractal dimension (FD) analysis in assessing structural alterations in coronary arterioles following 5/6 nephrectomy in rats, a widely used model for chronic kidney disease (CKD). CKD is associated with a heightened risk of cardiovascular morbidity, partly due to microvascular damage. Structural changes in coronary arterioles-typically 10-150 µm in diameter-such as medial thickening, increased collagen deposition, and lumen narrowing, can impair myocardial perfusion and contribute to adverse cardiovascular outcomes. To quantitatively characterize these microvascular changes, we employed FD analysis, a mathematical approach capable of evaluating tissue complexity and self-similarity. This methodology enables objective quantification of remodeling in coronary resistance vessels under pathological conditions. Our findings suggest that FD analysis serves as a reliable marker for the evaluation of microvascular integrity in CKD and provides insights into the mitigating effects of pharmacological interventions targeting the renin-angiotensin system.

Fluorescence microscopy (FM) utilizes fluorescent staining to label intra- and extracellular molecules, offering high-resolution imaging capabilities and facilitating the study of various pathological processes. This study aims to optimize staining and imaging protocols for peripheral blood mononuclear cells (PBMC) using FM, which is crucial for obtaining reliable and reproducible results. After optimization, FM could become one of the fundamental research techniques focused on understanding immune cell function. Despite extensive research, there is no standard PBMC staining protocol for FM, which still generates many issues with this technique's fast and proper usage. We aim to bridge this gap through systematic optimization of each step of the staining and imaging process. Our research includes selecting the most suitable fluorescent dyes, optimizing fixation and permeabilization protocols, and studying imaging parameters. The aim is to improve the signal-to-noise ratio and obtain high-resolution images of stained PBMC. By developing a standard methodology, this research may contribute to the advancement of FM-based techniques for PBMC, offering researchers more possibilities for exploring the immune system.

This investigation aims to extract cellulose from four distinct apple varieties-Golden Delicious, Granny Smith, Gala, and Red Delicious-and determine its structural characteristics at different scales using advanced techniques such as super-resolution microscopy and spectroscopy. The cellulose microfibrils (>1 µm diameter) consist of nanofibrils (<500 nm) containing 2D nanofibers (30-200 nm). The crystalline structure of cellulose in the four apple varieties presents a range of lattice spacing between 3.82 and 5.96 Å. This is measured by using microscopy and computational simulation to determine the corresponding crystalline structure from the d-spacings and angles between different crystalline planes in the crystalline regions of the different apple celluloses. It is shown that the triclinic phase is predominant in all cellulose types under investigation. At the same time, measurement of nanomechanical properties shows that the Young's modulus of cellulose is around 2.50-4.54 GPa. The novelty of this contribution is to demonstrate that the simulated unit cells differ from other reported types of cellulose because the unit cell depends upon the microstructural arrangement and the morphology of the different tissues, giving rise to distinct properties.

In cryo-transmission electron microscopy, single-particle reconstructions exploit the weak phase object approximation. A decisive aspect to be studied systematically is to what extent underlying scattering assumptions limit the resolution, whether theoretical limits are compatible with experimental observations, and if current experimental benchmarks achieve this limit. Single-, multislice, and hybrid scattering models are employed in this work for simulating eight protein complexes up to 97.5 nm in thickness, embedded in low-density amorphous ice obtained from molecular dynamics. With the multislice scheme providing an accurate solution to the multiple scattering problem as reference, the reliability of the different models is assessed in both real and Fourier space, particularly via Fourier ring correlations at the specimen exit wave level. A comparison with benchmarking literature resolutions is performed. Our results show proportionality of the attainable resolution to the square root of the projection thickness. This is in reasonable quantitative agreement with the highest resolution published experimentally for proteins with at least the size of apoferritin. The study provides a rationale for the expectable resolution for a protein complex of known size. The implications of structural noise due to the ice background for the minimal ice thickness on protein size-dependent resolution are discussed, as well as efficient methods to approximate multiple scattering and propagation in thick proteins.