low voltage electron microscopy
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Three-dimensional structure models refined using low-resolution data from crystallographic or electron cryo-microscopy experiments can benefit from high-quality restraints derived from quantum-chemical methods. However, nonperiodic atom-centered quantum-chemistry codes do not inherently account for nearest-neighbor interactions of crystallographic symmetry-related copies in a satisfactory way. Here, these nearest-neighbor effects have been included in the model by expanding to a super-cell and then truncating the super-cell to only include residues from neighboring cells that are interacting with the asymmetric unit. In this way, the fragmentation approach can adequately and efficiently include nearest-neighbor effects. It has previously been shown that a moderately sized X-ray structure can be treated using quantum methods if a fragmentation approach is applied. In this study, a target protein (PDB entry 4gif) was partitioned into a number of large fragments. The use of large fragments (typically hundreds of atoms) is tractable when a GPU-based package such as TeraChem is employed or cheaper (semi-empirical) methods are used. The QM calculations were run at the HF-D3/6-31G level. The models refined using a recently developed semi-empirical method (GFN2-xTB) were compared and contrasted. To validate the refinement procedure for a non-P1 structure, a standard set of crystallographic metrics were used. The robustness of the implementation is shown by refining 13 additional protein models across multiple space groups and a summary of the refinement metrics is presented.
The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.
Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post-fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512-517, 2016. © 2016 Wiley Periodicals, Inc.
- MeSH
- design vybavení MeSH
- elektronová mikroskopie přístrojové vybavení metody MeSH
- epoxidové pryskyřice chemie MeSH
- mikrotomie metody MeSH
- myokard ultrastruktura MeSH
- myši MeSH
- polymery chemie MeSH
- srdce diagnostické zobrazování MeSH
- zalévání tkání plastickou hmotou metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
... -- 3.3.3 Surface and adsorption phenomena 70 -- 3.3.4 Galvanic cell 72 -- 3.3.5 Resting membrane voltage ... ... (potential) 74 -- 3.3.5.1 The Nemst equation for resting membrane voltage 74 -- 3.3.5.2. ... ... The Goldman equation (resting membrane voltage) 80 -- 3.4.3 Nonlinear thermodynamics and dissipative ... ... microscopy 223 -- 11.6.2.1 Transmission electron microscopy 223 -- 11.6.2.2 Scanning electron microscopy ... ... (SEM) 224 -- 11.6.3 Acoustic microscopy 225 -- 11.7 Bone densitometry (I. ...
2nd rev. ed. 325 s. : il., tab. ; 30 cm
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- fyzika, biofyzika
- NLK Publikační typ
- učebnice vysokých škol
... -- Number and Geometry of Covalent Bonds It Can Make 33 -- CONTENTS -- XV -- Electrons May Be Shared ... ... 387 -- Q[ Electron Microscopy: Methods and Applications 388 -- Resolution of Transmission Electron Microscopy ... ... is -- Vastly Greater Than That of Light Microscopy 388 -- Cryoelectron Microscopy Allows Visualization ... ... of -- Particles Without Fixation or Staining 389 -- Electron Microscopy of Metal-Coated Specimens Can ... ... 494 -- Reduction Potentials of Electron Carriers Favor -- Electron Flow from NADH to 02 499 -- Experiments ...
6th ed. xxxvii, 1150 s. : il., tab. ; 29 cm
- MeSH
- biologie buňky MeSH
- molekulární biologie MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
- cytologie, klinická cytologie
Voltage-sensitive dyes (VSDs) are used to record transient potential changes in various cardiac preparations. In our laboratory, action potentials have been recorded by optical probe using di-4-ANEPPS. In this study, the effects of two different ways of staining were compared in guinea pig and rabbit isolated hearts perfused according to Langendorff: staining either by coronary perfusion with low dye concentration or with concentrated dye as a bolus into the aorta. Staining with low dye concentration lead to its better persistence in the tissue. Electrogram and coronary flow were monitored continuously. During the staining and washout of the dye, prominent electrophysiological changes occurred such as a decrease in spontaneous heart rate, partial atrioventricular block and changes of ST-T segment, accompanied by a decrease in mean coronary flow. No production of hydroxyl radicals was found by HPLC which excluded significant ischemic damage of the myocardium. Good viability of the stained preparation was supported by unchanged electron microscopy. Since in rabbit hearts the VSD-induced arrhythmogenesis was less pronounced, we conclude that the rabbit myocardium is more resistant to the changes triggered by VSD application. It may be due to different properties of the membrane potassium channels in the cardiomyocytes of these two species.
- MeSH
- akční potenciály MeSH
- barvení a značení MeSH
- draslíkové kanály metabolismus MeSH
- elektrofyziologie MeSH
- financování organizované MeSH
- fluorescenční barviva toxicita MeSH
- kardiomyocyty metabolismus účinky léků MeSH
- králíci MeSH
- morčata MeSH
- myokard metabolismus ultrastruktura MeSH
- perfuze MeSH
- pyridinové sloučeniny toxicita MeSH
- srdce patofyziologie účinky léků MeSH
- srdeční arytmie chemicky indukované patofyziologie MeSH
- techniky in vitro MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- morčata MeSH
- zvířata MeSH
... properties of particles and their consequences 5 -- 2.1.2 General properties of atoms 7 -- 2.1.2.1 The electron ... ... (potential) 66 -- 3.3.5.1 The Nemst equation for resting membrane voltage 66 -- 3.3.5.2. ... ... The Goldman equation (resting membrane voltage) 72 -- 3.4.3 Nonlinear thermodynamics and dissipative ... ... microscopy 215 -- 11.6.2.1 Transmission electron microscopy 216 -- 11.6.2.2 Scanning electron microscopy ... ... (SEM) 216 -- 11.6.3 Acoustic microscopy 217 -- 11.7 Bone densitometry (I. ...
1. vyd. viii, 317 s. : il. ; 30 cm
... .: Observation of biological material in low voltage transmission electron microscope 75 -- ABSTRACTS ... ... J Bartoš J Lichnovský V.: TUNEL. apostain, lamin b and annexin v methods in flow cytometry and microscopy ...
197 s. : il. ; 20 cm
