Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.
- MeSH
- flavinadenindinukleotid * MeSH
- mikroskopie fluorescenční multifotonová MeSH
- motilita spermií MeSH
- NADP * MeSH
- spermie účinky léků MeSH
- štěnice MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Butterfly wings have complex structure lending it several interesting properties. Coloration of the wing is one of the first things to encounter and the overall visual effect is in fact influenced by several factors. Chemical pigments set the base color of the wing, topographical structures on the wing scales cause color shift by interference and their arrangement into diffraction grating causes iridescence. The thin film interference can be attributed to microscopic ridges covering wing scales. Observation and calculation of the color shift on wings of Euploea mulciber species using Fourier transform of images obtained by atomic force microscopy is the focus of this article.
- MeSH
- Fourierova analýza MeSH
- křídla zvířecí ultrastruktura MeSH
- mikroskopie atomárních sil MeSH
- motýli fyziologie ultrastruktura MeSH
- pigmentace fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Quantification of the structure and composition of biomaterials using micro-CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three-dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro-CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro-CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro-CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro-CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open-source software for calibrating quantitative micro-CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three-dimensional fibrillar and porous structures in micro-CT scans.
Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.
- MeSH
- barvení a značení MeSH
- buněčné kultury MeSH
- kolagen typu I analýza genetika metabolismus MeSH
- kultivované buňky MeSH
- Leydigovy buňky chemie metabolismus MeSH
- prasata MeSH
- srdeční chlopně chemie cytologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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
The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella.
The blood group system AB0 is determined by the composition of terminal oligosaccharides on red blood cells. Thanks to this structural feature, these groups can be recognized by saccharide-recognizing compounds. Lectins are proteins that are able to reversibly bind saccharide structures. They generally occur as multimers and are known as hemagglutination agents. Hemagglutination is a process in which blood cells are cross-linked via multivalent molecules. Apart from lectins, hemagglutination can also be caused by antibodies or viruses. A hemagglutination assay is commonly used for the detection of multivalent molecules that recognize blood cells, in order to search for their sugar specificity. It is traditionally performed on a microtiter plate, where the lectin solution is serially diluted and the lowest concentration of lectin causing agglutination is detected. This experimental set-up is utilized further for testing lectin specificity via a hemagglutination inhibition assay. We have developed a new way of detecting hemagglutination using microscopy, which was tested on purified lectins as well as cell lysates. Hemagglutination was performed on a microscope slide directly and detected using a microscope. Comparison with the standard hemagglutination assay using microtiter plates revealed that microscopic approach is faster and more robust and allows fast determination of lectin activities immediately in bacterial cytosols.
- MeSH
- hemaglutinace * MeSH
- hemaglutinační testy metody MeSH
- hemolýza MeSH
- lektiny metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- testy inhibice hemaglutinace metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This article proposes an innovative methodology which employs nondestructive techniques to assess the effectiveness of new formulations based on ionic liquids, as alternative solvents for enzymes (proteases), for the removal of proteinaceous materials from painted surfaces during restoration treatments. Ionic liquids (ILs), also known as "designer" solvents, because of their peculiar properties which can be adjusted by selecting different cation-anion combinations, are potentially green solvents due totheir low vapour pressure. In this study, two ionic liquids were selected: IL1 (1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4 ])) and IL2 (1-ethyl-3-methylimidazolium ethylsulphate ([EMIM][EtSO4 ])). New formulations were prepared with these ILs and two different proteases (E): one acid (E1-pepsin) and one alkaline (E2-obtained from Aspergillus sojae). These formulations were tested on tempera and oil mock-up samples, prepared in accordance with historically documented recipes, and covered with two different types of protein-based varnishes (egg white and isinglass-fish glue). A noninvasive multiscale imaging methodology was applied before and after the treatment to evaluate the cleaning's effectiveness. Different microscopic techniques-optical microscopy (OM) with visible and fluorescent light, scanning electron microscopy (SEM) and atomic force microscopy (AFM)-together with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) were applied on areas cleaned with the new formulations (IL + E) and reference areas cleaned only with the commercial enzyme formulations (gels). MALDI-TOF proved particularly very useful for comparing the diversity and abundance of peptides released by using different enzymatic systems. Microsc. Res. Tech. 77:574-585, 2014. © 2014 Wiley Periodicals, Inc.
The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi-modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping-together artifact of the measurement or an inherent intra-cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra-cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM-algorithm-based method is presented for the conversion of multi-modal to quantal data that provides as by-product estimates of means and variances of basic granule packaging.
- MeSH
- biologie buňky statistika a číselné údaje MeSH
- cytoplazmatická granula chemie metabolismus MeSH
- interpretace statistických dat MeSH
- lidé MeSH
- proteiny metabolismus MeSH
- sekreční vezikuly chemie metabolismus MeSH
- synaptické vezikuly chemie metabolismus MeSH
- velikost částic MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Lurcher mutant mice represent a natural model of genetically-determined olivocerebellar degeneration caused by a mutation in the δ2 glutamate receptor gene. They suffer from progressive postnatal loss of cerebellar Purkinje cells and a decrease of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. A confocal laser scanning microscope was used aiming investigation the dynamics of changes in the cerebellar cortex of Lurcher and wild type mice derived from two strains during the period of 8-21 postnatal days. Fluorescent double-staining was used to visualize mainly the Purkinje cells in cerebellar slices. In wild types, only normal Purkinje cells of round or regular drop-shaped were present, when staining intensity of other individual cell structures differed in dependence on the age of the animal. In Lurcher mutants, there were still some normal-shaped cells. Nevertheless, depending on the animal's age, a wide variety of stages of the cell degeneration were depicted. The main characteristics of Purkinje cell degeneration in the early stage are: disruption of the continuity of the Purkinje cell layer, dark spots in cell nuclei and an irregular coloring of the cytoplasm. Later, the cells and their nuclei were deformed, often with two main dendrites sprouting from the cell body. Finally, the cell and nucleus margins were unclear, dendrites were significantly thickened, showing signs of shrinkage and fragmentation. Cell nucleoli underwent changes in number and appearance. No differences between the Lurcher mice of both strains (C3H and B6CBA) under examination were found.
- MeSH
- barvení a značení metody MeSH
- časové faktory MeSH
- fluorescenční barviva metabolismus MeSH
- glutamátové receptory nedostatek MeSH
- konfokální mikroskopie MeSH
- kůra mozečku patologie MeSH
- myši - mutanty neurologické MeSH
- myši MeSH
- Purkyňovy buňky chemie cytologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
