scanning electron microscopy (SEM)
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When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine.
Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.
- MeSH
- buněčná membrána ultrastruktura MeSH
- buněčné jádro ultrastruktura MeSH
- cytoskelet ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- rostlinné buňky ultrastruktura MeSH
- rostliny anatomie a histologie ultrastruktura MeSH
- tabák anatomie a histologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Testing the hemocompatibility of medical devices after their interaction with blood entails the need to evaluate the activation of blood elements and the degree of their coagulation and adhesion to the device surface. One possible way to achieve this is to use scanning electron microscopy (SEM). The aim was to develop a novel SEM-based method to assess the thrombogenic potential of medical devices and their adhesiveness to blood cells. As a part of this task, also find a convenient procedure of efficient and non-destructive sample fixation for SEM while reducing the use of highly toxic substances and shortening the fixation time. A polymeric surgical mesh was exposed to blood so that blood elements adhered to its surface. Such prepared samples were then chemically fixed for a subsequent SEM measurement; a number of fixation procedures were tested to find the optimal one. The fixation results were evaluated from SEM images, and the degree of blood elements' adhesion was determined from the images using ImageJ software. The best fixation was achieved with the May-Grünwald solution, which is less toxic than chemicals traditionally used. Moreover, manipulation with highly toxic osmium tetroxide can be avoided in the proposed procedure. A convenient methodology for SEM image analysis has been developed too, enabling to quantitatively evaluate the interaction of blood with the surfaces of various medical devices. Our method replaces the subjective assessment of surface coverage with a better-defined procedure, thus offering more precise and reliable results.
- MeSH
- histologické techniky * MeSH
- mikroskopie elektronová rastrovací MeSH
- oxid osmičelý * MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- adheziva MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací MeSH
- preparace zubní kavity MeSH
- trvalá zubní náhrada metody MeSH
- zubní materiály MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinické zkoušky MeSH
8 sv.
OBJECTIVE: Allograft heart valves (AHV), biological valves of human origin, offer potential advantages over conventional xenografts in terms of superior hemodynamics and, perhaps, better durability. The most important factors for long-term AHV clinical performance are the processing and cryopreservation methods. The aim of this study was to evaluate the impact of current processing protocol on valve tissue morphology, mainly to address the effect of successive processing steps on the leaflet surface structure. For the detection of fine changes in endothelial covering and underlying layers, our own modification of the scanning electron microscopy (SEM) technique was utilized. MATERIAL AND METHODS: The study was based on an investigation of 20 AHV (40 specimens). Fourteen valves came from heart-beating donors (multiorgan harvesting) when the heart could not be transplanted for any reason (donor criteria, availability of recipient and/or logistics). Six were obtained at the time of routine postmortems--non heart-beating donors (NHBD). All specimens were initially fixed in Baker's solution. Tissue samples were dissected, dried with hexamethyldisilazane (HMDS), gold-coated, studied and photographed by SEM (Tesla BS 301). In order to define the integrity of the endothelium, subendothelial layers and the quality of the surface under SEM, a special six-level score system was introduced: 1-intact endothelium, 2-confluent endothelium with structural inhomogeneity, 3-disruption of intercellular contacts, 4-separation of endothelial cells, 5-complete loss of endothelium, 6-damage of subendothelial layers). AHV samples were divided into 4 groups for comparison. One aortic AHV "fresh" control sample obtained from a heart-beating donor was evaluated without any processing and was compared with (i) tissue from AHV obtained from NHBD with warm ischemia of 12 and 48 hours, (ii) samples stored at +4 degrees C in saline for 24 h, (iii) antibiotic-treated tissue for 24 h at 37 degrees C and finally with (iv) cryopreserved valves stored in liquid nitrogen (-196 degrees C) for 6-38 months. RESULTS: Our alternative for drying samples by the HMDS method proved to be suitable for thin membranes of human semilunar valves. We were able to detect early changes in the endothelium after harvesting and denudation of the endothelial covering during preservation with and without freezing. The surface of the AHV samples revealed the typical features and score system determined endothelial cell damage. Control "fresh" sample: score 2, (i) NHBD samples with warm ischemia of 12 h: score 3-4, with warm ischemia of 48 h: score 4-5, (ii) samples stored at +4 degrees C in saline for 48 h: score 5-6, (iii) antibiotic-treated tissue for 24 h at 37 degrees C: score 5, (iv) cryopreserved valves stored in liquid nitrogen for 6-38 months: score 5-6. CONCLUSION: SEM (using HMDS drying) together with other methods may be helpful for the morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. Severe AHV leaflet endothelial destruction was proven on AHV grafts. These changes arose already in the initial steps of tissue processing, just after the donor heart harvesting and then at the time of antibiotic valve graft treatment. These results are considered as the starting point for the development of a better preservation protocol.
- MeSH
- bazální membrána patologie MeSH
- cévní endotel patologie MeSH
- financování organizované MeSH
- homologní transplantace MeSH
- kryoprezervace MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací MeSH
- odběr tkání a orgánů MeSH
- povrchové vlastnosti MeSH
- srdeční chlopně patologie transplantace MeSH
- teplá ischemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
Urolithiasis is a frequent and in many cases serious disease. Proper analysis of kidney stone composition is crucial for appropriate treatment and prevention of disease recurrence. In this work, scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy was applied for a study of 30 samples covering the most common types of human kidney stones. The results are analyzed and evaluated in terms of applicability of the method for both routine kidney stone analysis as well as collecting of specific data. The method provides complex information about studied samples including morphology of the stones and of the present crystals or their aggregates. It also brings information on elemental composition of the phases. After application of standardization, quantitative microanalysis with detection limits of 400 ppm (Mg, P, S, Cl, K, Ca), 500 ppm (Na) and 1200 ppm (F) was obtained. Compositional mapping with EDS shows the elemental distribution within a sample. This study demonstrated that information on morphology and chemistry acquired by these methods was highly reliable for identification of phases, even when present in small amounts. It provided information on kidney stone structure, relationships between phases, major and minor element content, and variations in chemical composition related to the growth of the stones. SEM represents a powerful tool in urinary stone analysis, since a single facility can produce a wide spectrum of information. It can be suggested as a basic method used for routine urinary stone identification, whilst bringing additional detailed information that cannot be obtained by other methods.
