Atherosclerotic alterations in human carotid observed by scanning electron microscopy.

Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal portion are fundamental to understand the evolution and gravity of lesions. This study shows that scanning microscopy is instrumental for better understanding the physiopathology of this disease

DIY adapting SEM for low-voltage TEM imaging

Electron microscopy is essential for examining materials and biological samples at microscopic levels, providing detailed insights. Achieving high-quality imaging is often challenged by the potential damage high-energy beams can cause to sensitive samples. This study compares scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to evaluate image quality, noise levels, and the ability to preserve delicate specimens. We used a modified SEM system with a transmitted electrons conversion accessory, allowing it to operate like a TEM but at lower voltages, thereby reducing sample damage. Our analysis included quantitative assessments of noise levels and texture characteristics such as entropy, contrast, dissimilarity, homogeneity, energy, and correlation. This comprehensive evaluation directly compared traditional TEM and the adapted SEM system across various images. The results showed that TEM provided images with higher clarity and significantly lower noise levels, reinforcing its status as the preferred method for detailed studies. However, the modified SEM system also produced high-quality images at very low acceleration voltages, which is crucial for imaging samples sensitive to high-energy exposure. The texture metrics analysis highlighted the strengths and limitations of each method, with TEM images exhibiting lower entropy and higher homogeneity, indicating smoother and more uniform textures. This study emphasizes the importance of selecting the appropriate electron microscopy method based on research needs, such as sample sensitivity and required detail level. With its conversion accessory, the modified SEM system is a versatile and valuable tool, offering a practical alternative to TEM for various applications. This research enhances our understanding of the capabilities and limitations of SEM and TEM. It paves the way for further innovations in electron microscopy techniques, improving their applicability for studying sensitive materials. Research Highlights: Our study introduces a modified SEM adapter enabling TEM-like imaging at reduced voltages, effectively minimizing sample damage without compromising image resolution. Through comparative analysis, we found that images from the modified SEM closely match the quality of traditional TEM, showcasing significantly lower noise levels. This advancement underscores the SEM's enhanced capability for detailed structural analysis of sensitive materials, broadening its utility across materials science and biology. © 2024 The Author(s). Microscopy Research and Technique published by Wiley Periodicals LL

The synovial surface of the articular cartilage

The articular cartilage has been the subject of a huge amount of research carried out with a wide array of different techniques. Most of the existing morphological and ultrastructural data on the this tissue, however, were obtained either by light microscopy or by transmission electron microscopy. Both techniques rely on thin sections and neither allows a direct, face-on visualization of the free cartilage surface (synovial surface), which is the only portion subject to frictional as well as compressive forces. In the present research, high resolution visualization by scanning electron microscopy and by atomic force microscopy revealed that the collagen fibrils of the articular surface are exclusively represented by thin, uniform, parallel fibrils evocative of the heterotypic type IX-type II fibrils reported by other authors, immersed in an abundant matrix of glycoconjugates, in part regularly arranged in phase with the D-period of collagen. Electrophoresis of fluorophore-labeled saccharides confirmed that the superficial and the deeper layers are quite different in their glycoconjugate content as well, the deeper ones containing more sulfated, more acidic small proteoglycans bound to thicker, more heterogenous collagen fibrils. The differences found between the synovial surface and the deeper layers are consistent with the different mechanical stresses they must withstand

Ultrastructural Aspects of Physiological Mineralization: A Comparative Study in Different Hard Tissues

The calcified tissues of vertebrates are essentially represented by bone, cartilage, dentin and calcified tendons. In all these tissues a major hallmark of mineralization is the deposition of the inorganic phase on a pre-existing collagen template, but evident differences exist among these materials and the molecular details of the process are still incompletely understood. In this study, the ultrastructural aspects of the mineral phase of these tissues were investigated by means of high-resolution scanning electron microscopy (HR-SEM) after low-temperature thermal deproteination, a technique allowing a direct, unrestricted visualization of the mineral component. Each tissue showed distinctive features. In most cases, calcification proceeds in a discontinuous way through the formation of clumps or clusters of mineralized tissue; in all cases, except cartilage, the mineral phase shows an evident relationship with the layout and/or the D-period of the collagen fibrils. Our results highlight the peculiar aspect of the mineralization process in the cartilage with respect to the other tissues, all of them containing collagen type I instead of type II, and suggest that a different molecular mechanism may be at work. It is still unclear whether and how this may be related to the content, exclusive of cartilage, of collagen type II. The identification of the tissue-specific features exhibited by cartilage versus those shared by all the other three tissues, although from different species, requires further research on physiological calcification

Mitochondria as target to inhibit proliferation and induce apoptosis of cancer cells: the effects of doxycycline and gemcitabine

Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the adenocarcinoma cell line A549, its mitochondrial DNA-less derivative A549 ρ° and cultured fibroblasts. Treatment with doxycycline for 5 days resulted in a decrease of mitochondrial-encoded proteins, respiration and membrane potential, and an increase of reactive oxygen species in A549 cells and fibroblasts, but fibroblasts were less affected. Doxycycline slowed proliferation of A549 cells by 35%. Cellular ATP levels did not change. Doxycycline alone had no effect on apoptosis; however, in combination with gemcitabine given during the last 2 days of treatment, doxycycline increased caspase 9 and 3/7 activities, resulting in a further decrease of surviving A549 cells by 59% and of fibroblasts by 24% compared to gemcitabine treatment alone. A549 ρ° cells were not affected by doxycycline. Key effects of doxycycline observed in A549 cells, such as the decrease of mitochondrial-encoded proteins and surviving cells were also seen in the cancer cell lines COLO357 and HT29. Our results suggest that doxycycline suppresses cancer cell proliferation and primes cells for apoptosis by gemcitabine

Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration

The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease

Mitochondria as oncotarget: a comparison between the tetracycline analogs doxycycline and COL-3

Tetracyclines have anticancer properties in addition to their well-known antibacterial properties. It has been proposed that tetracyclines slow metastasis and angiogenesis through inhibition of matrix metalloproteinases. However, we believe that the anticancer effect of tetracyclines is due to their inhibition of mitochondrial protein synthesis, resulting in a decrease of the mitochondrial energy generating capacity. Several groups have developed analogs that are void of antibacterial action. An example is COL-3, which is currently tested for its anticancer effects in clinical trials. We have undertaken a comparative study of the tetracycline analogs COL-3 and doxycycline, which has an antibacterial function, to further investigate the role of the mitochondrial energy generating capacity in the anticancer mechanism and, thereby, evaluate the usefulness of mitochondria as an oncotarget. Our experiments with cultures of the human A549, COLO357 and HT29 cancer cells and fibroblasts indicated that COL-3 is significantly more cytotoxic than doxycycline. Mitochondrial translation assays demonstrated that COL-3 has retained its inhibitory effect on mitochondrial protein synthesis. Both drugs caused a severe decrease in the levels of mitochondrially encoded cytochrome-c oxidase subunits and cytochrome-c oxidase activity. In addition, COL-3 produced a marked drop in the level of nuclear-encoded succinate dehydrogenase subunit A and citrate synthase activity, indicating that COL-3 has multiple inhibitory effects. Contrary to COL-3, the anticancer action of doxycycline appears to be based specifically on inhibition of mitochondrial protein synthesis, which is thought to affect rapidly proliferating cancer cells more than healthy tissue. Doxycycline is likely to cause less side effects that COL-3

Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration.

The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease

The collagenic architecture of human dura mater: Laboratory investigation

Object. Human dura mater is the most external meningeal sheet surrounding the CNS. It provides an efficient protection to intracranial structures and represents the most important site for CSF turnover. Its intrinsic architecture is made up of fibrous tissue including collagenic and elastic fibers that guarantee the maintenance of its biophysical features. The recent technical advances in the repair of dural defects have allowed for the creation of many synthetic and biological grafts. However, no detailed studies on the 3D microscopic disposition of collagenic fibers in dura mater are available. The authors report on the collagenic 3D architecture of normal dura mater highlighting the orientation, disposition in 3 dimensions, and shape of the collagen fibers with respect to the observed layer. Methods. Thirty-two dura mater specimens were collected during cranial decompressive surgical procedures, fixed in 2.5% Karnovsky solution, and digested in 1 N NaOH solution. After a routine procedure, the specimens were observed using a scanning electron microscope. Results. The authors distinguished the following 5 layers in the fibrous dura mater of varying thicknesses, orientation, and structures: bone surface, external median, vascular, internal median, and arachnoid layers. Conclusions. The description of the ultrastructural 3D organization of the different layers of dura mater will give us more information for the creation of synthetic grafts that are as similar as possible to normal dura mater. This description will be also related to the study of the neoplastic invasion