Transmission Electron Microscopy of Biological Samples

During the last 70 years, transmission electron microscopy (TEM) has developed our knowledge about ultrastructure of the cells and tissues. Another aim is the determination of molecular structure, interactions and processes including structure-function relationships at cellular level using a variety of TEM techniques with resolution in atomic to nanometre range. Even with the best transmission electron microscope, it is impossible to obtain real results without optimal sample preparation, respecting both the structure and the antigenicity preservation. Preparation techniques for high-resolution study of both macromolecular complex and organelles within cellular complex are based on fast cryoimmobilisation process, where the sample is in the most native, hydrated state. Next, thin samples are directly visualised under cryo-transmission electron microscopy (cryo-TEM), while thicker samples require a thinning step via cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-focused ion beam (cryo-FIB) before visualisation. Alternatively, vitrified samples are freeze substituted and embedded in chosen resin for room temperature ultramicrotomy. This preparation technique is suitable for morphological study, 3D analysis of cellular interior and immunoelectron microscopy. A different route for immunolocalisation study is cryosectioning according to the Tokuyasu technique that is a choice for rare or methacrylate-sensitive antigens. Most recently, new hybrid techniques have been developed for difficult-to-fix organisms and antigens or labile and anoxia-sensitive tissues. Another preparation technique is, the oldest but still important, conventional chemical fixation dedicated in a wide range of research interest, involving morphological and immunolocalisation study. In this chapter, we present different sample preparation approaches for transmission electron microscopy of biological samples, including its methodological basis and applications

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique.(Folia Histochemica et Cytobiologica 2014, Vol. 52, No, 1, 1–17

Evaluation of leaf area index of winter wheat canopy by means of ground-based stereoscopic vision

audience: researcher, professiona

A different ultrastructural face of ribbon synapses in the rat retina

Abstract Ribbon synapses located exclusively within retinal, cochlear and vestibular connections belong to the most interesting cellular structures but their molecular nature and functions had remained unclear. The study has provided a descriptive morphological analysis of rat eye ribbon synapses using high-resolution transmission electron microscopy (TEM). An original collection of untypical, rarely present in the literature sagittal or tangential sections through the single RIBEYE domain of the particular ribbon have been delivered

Phage Therapy:What Have We Learned?

In this article we explain how current events in the field of phage therapy may positively influence its future development. We discuss the shift in position of the authorities, academia, media, non-governmental organizations, regulatory agencies, patients, and doctors which could enable further advances in the research and application of the therapy. In addition, we discuss methods to obtain optimal phage preparations and suggest the potential of novel applications of phage therapy extending beyond its anti-bacterial action

Phage Therapy in Prostatitis: Recent Prospects

Prostatitis has various etiology including bacterial infection and dysregulated immunity; some of its forms remain a serious therapeutic challenge. Inflammation occurs in all forms of this disorder and is proposed to predispose to the development of prostate cancer (PC). There are reports that phage therapy is effective in chronic bacterial prostatitis. Recent findings suggest that phages not only eliminate bacteria, but also mediate immunomodulating (for example, anti-inflammatory) functions. The immunomodulating effects of phages could be beneficial in treating all forms of prostatitis and play some role in the prevention of the development of PC. As the etiological factors contributing to the majority of prostatitis cases remains largely unknown, and management options are often likewise limited, phage therapy merits further research as an attractive therapeutic option given its immunomodulating effects irrespective of the underlying causative factor(s)

Rola diety w terapii tocznia rumieniowatego układowego

Toczeń rumieniowaty układowy (SLE) jest chorobą autoimmunologiczną, której częstość występowania rośnie. Z aktywnością SLE oraz samopoczuciem chorych może się wiązać dieta. Składnikiem odżywczym o najlepiej udokumentowanym działaniu w tym zakresie są kwasy omega-3. Wykazano, że ich suplementacja zmniejsza aktywność choroby. Uzupełnienie niedoborów witaminy D u chorych z SLE redukuje uczucie zmęczenia oraz odgrywa istotną rolę w prewencji osteoporozy. Ogromne znaczenie ma również utrzymanie prawidłowej masy ciała. Redukcja nadmiernej masy ciała wiąże się z niższym ryzykiem powikłań i zmniejsza uczucie zmęczenia. Dlatego chorzy z SLE powinni stosować zbilansowaną kalorycznie dietę, bogatą w warzywa i owoce oraz opierającą się na zasadach diety śródziemnomorskiej. Chorzy z powikłaniami, takimi jak choroby układu sercowo-naczyniowego, toczniowe zapalenie nerek prowadzące do ich przewlekłej niewydolności oraz osteoporoza, stanowią grupę, która wymaga szczególnej opieki dietetyka. Niestety, rolę interwencji dietetycznej w tych grupach chorych oceniano w niewielu badaniach. Strategie postępowania dietetycznego w SLE ograniczają się zatem do ogólnych wytycznych dotyczących modyfikacji czynników ryzyka chorób układu sercowo-naczyniowego oraz suplementacji witaminy D i wapnia w celu zmniejszenia ryzyka osteoporozy. Dopóki nie pojawią się szczegółowe zalecenia dietetyczne dla chorych z SLE, zasadne jest stosowanie ogólnych rekomendacji dotyczących profilaktyki chorób układu sercowo-naczyniowego, dyslipidemii, osteoporozy i zapobiegania progresji przewlekłej choroby nerek

The Role of Antibiotic Resistant A. baumannii in the Pathogenesis of Urinary Tract Infection and the Potential of Its Treatment with the Use of Bacteriophage Therapy

Acinetobacter baumannii are bacteria that belong to the critical priority group due to their carbapenems and third generation cephalosporins resistance, which are last-chance antibiotics. The growing multi-drug resistance and the ability of these bacteria to form biofilms makes it difficult to treat infections caused by this species, which often affects people with immunodeficiency or intensive care unit patients. In addition, most of the infections are associated with catheterization of patients. These bacteria are causative agents, inter alia, of urinary tract infections (UTI) which can cause serious medical and social problems, because of treatment difficulties as well as the possibility of recurrence and thus severely decrease patients’ quality of life. Therefore, a promising alternative to standard antibiotic therapy can be bacteriophage therapy, which will generate lower costs and will be safer for the treated patients and has real potential to be much more effective. The aim of the review is to outline the important role of drug-resistant A. baumannii in the pathogenesis of UTI and highlight the potential for fighting these infections with bacteriophage therapy. Further studies on the use of bacteriophages in the treatment of UTIs in animal models may lead to the use of bacteriophage therapy in human urinary tract infections caused by A. baumannii in the future

Animal Models in the Evaluation of the Effectiveness of Phage Therapy for Infections Caused by Gram-Negative Bacteria from the ESKAPE Group and the Reliability of Its Use in Humans

The authors emphasize how extremely important it is to highlight the role played by animal models in an attempt to determine possible phage interactions with the organism into which it was introduced as well as to determine the safety and effectiveness of phage therapy in vivo taking into account the individual conditions of a given organism and its physiology. Animal models in which phages are used make it possible, among other things, to evaluate the effective therapeutic dose and to choose the possible route of phage administration depending on the type of infection developed. These results cannot be applied in detail to the human body, but the knowledge gained from animal experiments is invaluable and very helpful. We would like to highlight how useful animal models may be for the possible effectiveness evaluation of phage therapy in the case of infections caused by gram-negative bacteria from the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species) group of pathogens. In this review, we focus specifically on the data from the last few years

Human β-Defensin 2 and Its Postulated Role in Modulation of the Immune Response

Studies described so far suggest that human β-defensin 2 is an important protein of innate immune response which provides protection for the human organism against invading pathogens of bacterial, viral, fungal, as well as parasitical origin. Its pivotal role in enhancing immunity was proved in infants. It may also be considered a marker of inflammation. Its therapeutic administration has been suggested for maintenance of the balance of systemic homeostasis based on the appropriate composition of the microbiota. It has been suggested that it may be an important therapeutic tool for modulating the response of the immune system in many inflammatory diseases, offering new treatment modalities. For this reason, its properties and role in the human body discussed in this review should be studied in more detail