TEM-analysis
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Electron tomographic reconstructions suffer from a number of artefacts arising from effects accompanying the processes of acquisition of a set of tilted projections of the specimen in a transmission electron microscope and from its subsequent computational handling. The most pronounced artefacts usually come from imprecise projection alignment, distortion of specimens during tomogram acquisition and from the presence of a region of missing data in the Fourier space, the "missing wedge". The ray artefacts caused by the presence of the missing wedge can be attenuated by the angular image filter, which attenuates the transition between the data and the missing wedge regions. In this work, we present an analysis of the influence of angular filtering on the resolution of averaged repetitive structural motives extracted from three-dimensional reconstructions of tomograms acquired in the single-axis tilting geometry.
The limited specimen tilting range that is typically available in electron tomography gives rise to a region in the Fourier space of the reconstructed object where experimental data are unavailable - the missing wedge. Since this region is sharply delimited from the area of available data, the reconstructed signal is typically hampered by convolution with its impulse response, which gives rise to the well-known missing wedge artefacts in 3D reconstructions. Despite the recent progress in the field of reconstruction and regularization techniques, the missing wedge artefacts remain untreated in most current reconstruction workflows in structural biology. Therefore we have designed a simple Fourier angular filter that effectively suppresses the ray artefacts in the single-axis tilting projection acquisition scheme, making single-axis tomographic reconstructions easier to interpret in particular at low signal-to-noise ratio in acquired projections. The proposed filter can be easily incorporated into current electron tomographic reconstruction schemes.
- MeSH
- artefakty MeSH
- Fourierova analýza MeSH
- krysa rodu rattus MeSH
- líska ultrastruktura MeSH
- mozeček ultrastruktura MeSH
- počítačové zpracování obrazu * MeSH
- poměr signál - šum MeSH
- pyl ultrastruktura MeSH
- tomografie elektronová metody MeSH
- Trypanosoma brucei brucei ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline. The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project. The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.
According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA. However, arrangement of replisomes in other eukaryotic organisms including vertebrate cells is largely unknown. Here, we used in vivo labeling of DNA segments in combination with the electron microscopy tomography to describe the organization of replisomes in human HeLa cells. The experiments were devised in order to distinguish between a model of independent replisomes and a model of replisome couples. The comparative analysis of short segments of replicons labeled in pulse-chase experiments of various length shows that replisomes in HeLa cells are organized into the couples during DNA replication. Moreover, our data enabled to suggest a new model of the organization of replicated DNA. According to this model, replisome couples produce loop with the associated arms in the form of four tightly associated 30nm fibers.
- MeSH
- bromodeoxyuridin metabolismus MeSH
- buněčné jádro metabolismus ultrastruktura MeSH
- chromatin fyziologie ultrastruktura MeSH
- deoxyuracilnukleotidy metabolismus MeSH
- DNA-dependentní DNA-polymerasy chemie metabolismus MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- modely genetické MeSH
- multienzymové komplexy chemie metabolismus MeSH
- počítačové zpracování obrazu MeSH
- replikace DNA fyziologie MeSH
- replikon genetika MeSH
- tomografie elektronová MeSH
- Check Tag
- lidé MeSH
Cryo Electron Tomography (cryoET) plays an essential role in Structural Biology, as it is the only technique that allows to study the structure of large macromolecular complexes in their close to native environment in situ. The reconstruction methods currently in use, such as Weighted Back Projection (WBP) or Simultaneous Iterative Reconstruction Technique (SIRT), deliver noisy and low-contrast reconstructions, which complicates the application of high-resolution protocols, such as Subtomogram Averaging (SA). We propose a Progressive Stochastic Reconstruction Technique (PSRT) - a novel iterative approach to tomographic reconstruction in cryoET based on Monte Carlo random walks guided by Metropolis-Hastings sampling strategy. We design a progressive reconstruction scheme to suit the conditions present in cryoET and apply it successfully to reconstructions of macromolecular complexes from both synthetic and experimental datasets. We show how to integrate PSRT into SA, where it provides an elegant solution to the region-of-interest problem and delivers high-contrast reconstructions that significantly improve template-based localization without any loss of high-resolution structural information. Furthermore, the locality of SA is exploited to design an importance sampling scheme which significantly speeds up the otherwise slow Monte Carlo approach. Finally, we design a new memory efficient solution for the specimen-level interior problem of cryoET, removing all associated artifacts.
- MeSH
- algoritmy MeSH
- elektronová kryomikroskopie metody MeSH
- makromolekulární látky chemie MeSH
- metoda Monte Carlo MeSH
- počítačové zpracování obrazu metody MeSH
- reprodukovatelnost výsledků MeSH
- ribozomy chemie MeSH
- stochastické procesy * MeSH
- tomografie elektronová metody MeSH
- zobrazování trojrozměrné metody MeSH
- Publikační typ
- časopisecké články MeSH
Extracellular vesicles (EVs) function as important conveyers of information between cells and thus can be exploited as drug delivery systems or disease biomarkers. Transmission electron microscopy (TEM) remains the gold standard method for visualisation of EVs, however the analysis of individual EVs in TEM images is time-consuming if performed manually. Therefore, we present here a software tool for computer-assisted evaluation of EVs in TEM images. TEM ExosomeAnalyzer detects EVs based on their shape and edge contrast criteria and subsequently analyses their size and roundness. The software tool is compatible with common negative staining protocols and isolation methods used in the field of EV research; even with challenging TEM images (EVs both lighter and darker than the background, images containing artefacts or precipitated stain, etc.). If the fully-automatic analysis fails to produce correct results, users can promptly adjust the detected seeds of EVs as well as their boundaries manually. The performance of our tool was evaluated for three different modes with variable levels of human interaction, using two datasets with various heterogeneity. The semi-automatic mode analyses EVs with high success rate in the homogenous dataset (F1 score 0.9094, Jaccard coefficient 0.8218) as well as in the highly heterogeneous dataset containing EVs isolated from cell culture medium and patient samples (F1 score 0.7619, Jaccard coefficient 0.7553). Moreover, the extracted size distribution profiles of EVs isolated from malignant ascites of ovarian cancer patients overlap with those derived by cryo-EM and are comparable to NTA- and TRPS-derived data. In summary, TEM ExosomeAnalyzer is an easy-to-use software tool for evaluation of many types of vesicular microparticles and is available at http://cbia.fi.muni.cz/exosome-analyzer free of charge for non-commercial and research purposes. The web page contains also detailed description how to use the software tool including a video tutorial.
- Publikační typ
- časopisecké články MeSH
Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.
- Publikační typ
- časopisecké články MeSH
Dual-energy X-ray absorptiometry (DXA) is rapidly becoming more accessible and popular as a technique to monitor body composition. The reliability of DXA has been examined extensively using a number of different methodological approaches. This study sets up to investigate the accuracy of measuring the parameters of body composition (BC) by means of the whole-body and the segmental DXA method analysis with the typical error of measurement (TEM) that allows for expressing the error in the units of measure. The research was implemented in a group of 63 participants, all of whom were university students. Thirty-eight males (22.6±2.9 years, average body mass 77.5±8.4 kg) and 25 females (21.4±2.0 years, average body mass 58.6±7.2 kg) were recruited. The measured parameters included body mass (BM), fat-free mass (FFM), body fat (BF), bone mineral content (BMC), bone mineral density (BMD). For the whole-body analysis, the determined TEM was: BM at the level of 0.12 kg in females and 0.29 kg in males; BF 0.25kg and 0.44% females, 0.52 kg and 0.66% males; FFM 0.24 kg females and 0.42 kg males; BMC 0.02 kg females and males; BMD 0.01g/cm2 females and males. The TEM values in the segmental analysis were: BF within the range of 0.04-0.28 kg and 0.68-1.20% in females, 0.10-0.36 kg and 0.72-1.94% in males; FFM 0.08-0.41 kg females and 0.17-0.86 males, BMC 0.00-0.02 kg females and 0.01-0.02 kg males in relation to the body segment (upper limb, trunk, lower limb). The BMD value was at the level of 0.01-0.02g/cm2. The study results showed high reliability in measuring body composition parameters using the DXA method. The whole-body analysis showed a higher accuracy of measurement than the segmental. Only the changes that are greater than the TEM, or the upper bound (95%) of the confidence interval of the measurement can be considered demonstrable when interpreting repeated measurements.
- MeSH
- absorpční fotometrie metody MeSH
- dolní končetina diagnostické zobrazování MeSH
- dospělí MeSH
- horní končetina diagnostické zobrazování MeSH
- kostní denzita fyziologie MeSH
- lidé MeSH
- mladý dospělý MeSH
- reprodukovatelnost výsledků MeSH
- složení těla fyziologie MeSH
- studenti statistika a číselné údaje MeSH
- tělesná konstituce fyziologie MeSH
- tuková tkáň diagnostické zobrazování MeSH
- univerzity MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stable Pd nanocubes (PdNC) with the average size ~15 nm were prepared by the controlled reduction of sodium tetrachloropalladate with ascorbic acid in water, in the presence of polyvinylpyrrolidone and potassium bromide. Morphology of the particles was characterized by transmission electron microscopy (TEM) and their stability in the colloidal solution was verified by dynamic light scattering (DLS). It has been demonstrated that the Pd nanocubes can be distinguished from commercial Au nanospheres in a standard TEM microscope by means of automated image analysis. In the next step, the PdNC were successfully conjugated to immunoglobulin proteins and used for the detection of a specific protein (nucleophosmin) on ultrathin sections of HeLa cells. Our experiments showed that PdNC can be used for multiple immunolabeling in combination with commercial Au nanospheres.
- MeSH
- barvení a značení metody MeSH
- bromidy chemie MeSH
- HeLa buňky MeSH
- imunoglobuliny chemie MeSH
- imunokonjugáty chemie MeSH
- jaderné proteiny analýza MeSH
- koloidy MeSH
- kovové nanočástice chemie ultrastruktura MeSH
- kyselina askorbová chemie MeSH
- lidé MeSH
- mikrotomie MeSH
- palladium chemie MeSH
- počítačové zpracování obrazu MeSH
- povidon chemie MeSH
- sloučeniny draslíku chemie MeSH
- transmisní elektronová mikroskopie MeSH
- velikost částic MeSH
- voda MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cieľ práce: Cieľom štúdie bolo stanovenie prevalencie ESBL-pozitívnych izolátov Klebsielh pneumoniae u pacientov v intenzívnej starostlivosti a ich molekulárno-biologická analýza. Materiál a metódy: V období 5 mesiacov boli od pacientov hospitalizovaných na Klinike anestéziológie a resuscitácie Fakultnej nemocnice Olomouc izolované kmene Klehsiella pneumoniae. U každého izolátu bol určený antibiogram štandardnou dilučnou mikrometódou a produkcia ESBL bola stanovená modifikovaným double disk synergy testom. Na dôkaz prítomnosti génu blaTEM a blaSHV bola použitá PCR. Izoláty produkujúce SHV a TEM typy β-laktamáz boh ďalej typizované použitím metódy polymorfizmu dĺžky restrikčných fragmentov (RFLP) na identifikáciu najbežnejšie sa vyskytujúcich mutácií zodpovedných za vznik ESBL fenotypu. Posúdenie podobnosti, resp. identity izolátov, bolo uskutočnené pulznou gelovou elektroforézou (PFGE) fragmentov DNA, naštiepených pomocou reštrikčnej endonukleázy Xbal. Výsledky: Celkovo bolo získaných 67 izolátov Klebsiella pneumoniae. U 13 z nich bola stanovená produkcia ESBL a pomocou PCR dokázaná pritomnost WasHv génu. Reštrikčné štiepenie pomocou Nhel odhalilo výskyt mutácie v pozícii 238 u všetkých SHV-pozitívnych PCR produktov. Prítomnosť génu kódujúceho širokospektrú β-laktamázu TEM typu však nebola potvrdená. Molekulárno-biologická typizácia pomocou PFGE zistila prítomnosť 11 rôznych kmeňov. Záver. Prevalencia ESBL-pozitívnych kmeňov KlebsieJJa pneumoniae dosiahla u sledovanej skupiny pacientov v intenzívnej starostlivosti hodnoty 19,4 %. Analýza SHV a TEM produktov PCR metódou RFLP preukázala výskyt ESBL typu SHV. Celkovo 84,6 % kmenov malo jedinečný reštrikčný profil. Výsledky dokladujú nielen dobrú úroveň hygienicko-epidemiologických režimov na sledovanej klinike, ale aj racionálnu antibiotickú politiku.
Objectives: The study aimed at the assessment of the prevalence of ESBL-positive isolates of Klebsiella pneumoniae in intensive care patients and their molecular biology analysis. Material and methods: Over a 5-month period, Klebsiella pneumoniae strains were isolated from patients hospitalized at the Department of Anaesthesiology and Resuscitation of the University Hospital in Olomouc. For each isolate, an antibiogram was performed by the standard microdilution method and the production of ESBL was determined by the modified double-disk synergy test. PCR was used to demonstrate the presence of the blaTEM and blaSHV genes. The isolates producing SHV- and TEM-types of β-lactamases were typed using the restriction fragment length polymorphism (RFLP) method to identify the most common mutations responsible for the development of an ESBL phenotype. Similar or identical isolates were determined by pulsed-field gel electrophoresis (PFGE) of DNA fragments cleaved by the XbaI restriction endonuclease. Results: A total of 67 isolates of KJebsieJJa pneumoniae were obtained. In 13 of them, the production of ESBL was detected and the presence of the blaSHV gene was confirmed by PCR. Restriction cleavage by Nhei revealed mutations at position 238 in all SHV-positive PCR products. The restriction analysis did not confirm the presence of the gene encoding TEM-type extended-spectrum β-lactamase. Molecular biology typing by PFGE detected the presence of 11 different strains. Conclusions: In the observed group of intensive care patients, the prevalence of ESBL-positive strains of Klebsiella pneumoniae reached 19.4 %. The analysis of SHV and TEM products of PCR by the RFLPP method showed the prevalence of SHV-type ESBL. Overail, 84.6 % of the strains had unique restriction profiles. The results suggest both high levels of hygienic and epidemiological measures at the monitored department and rational antibiotic policy.
