atomic force microscopy (AFM) Dotaz Zobrazit nápovědu
Cíl práce: Oxidační stres je jedním z významných faktorů poškozujících spermie. Nadměrná produkce kyslíkových radikálů vede k poškození buněčné membrány a DNA. Cílem studie bylo využití AFM (Atomic Force Microscopy, mikroskopie atomárních sil) pro zobrazení povrchového poškození spermií vystavených experimentálnímu oxidačnímu stresu. Materiál a metodika: Byly získány a standardně zpracovány vzorky spermatu dárců. Pro vyšetření byl použit AFM systém Ntegra Vita (NT-MDT, Moskva, Rusko) s rozlišením v nanometrech. Po zobrazení spermií byly spermie exponovány v roztoku peroxidu vodíku (50 mmol/l) po 30 minut, po tuto dobu byly kontinuálně zobrazovány a ukládány scany zaznamenávající průběh děje v reálném čase. Následně byly vyhodnoceny a zaznamenány defekty povrchu. Výsledky: Po optimalizaci experimentálních podmínek byly zobrazeny morfologicky normální spermie a nalezeny defekty vyvolané peroxidem vodíku. Byla patrná ultrastruktura povrchu hlavičky v oblasti akrozomu. Závěr: Defekty spermie vyvolané experimentálním oxidačním stresem byly detailně zobrazeny. Studie potvrdila schopnost AFM techniky zobrazit morfologii spermií až s nanometrickým rozlišením. Tato metoda může být důležitým nástrojem pro výzkum oxidačního stresu a poznání jeho vlivu na snížení plodnosti. Příprava vzorku a nastavení přístroje vyžaduje optimalizaci několika parametrů.
- Klíčová slova
- neplodnost,
- MeSH
- financování organizované MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- mužská infertilita MeSH
- oxidační stres MeSH
- spermie patologie ultrastruktura MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
The aim of the present study was to evaluate the efficiency of photosensitisation induced by two photosensitizers, TMPyP and ClAlPcS2, tested in vitro on the tumor cell line MCF7. The oxidative damage of DNA in MCF-7 cells was analyzed by comet assay (CA) combined with Atomic Force Microscopy (AFM). The ability of detection of apoptotic response detected by Atomic Force Microscopy at the individual molecule level of DNA was successfully demonstrated; when DNA get damaged, cleavage to fragments caused by photodynamic treatment was directly visualized by AFM imaging of individual molecules. Its accuracy and reliability was validated through the comparison with traditional single cell agarose electrophoresis.
Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip-sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase-distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever-sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.
- MeSH
- endoteliální buňky ultrastruktura MeSH
- epitelové buňky MeSH
- eukaryotické buňky MeSH
- lidé MeSH
- mikroskopie atomárních sil metody MeSH
- myokard cytologie MeSH
- myši MeSH
- plíce cytologie MeSH
- pružnost MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.
- MeSH
- biomechanika * MeSH
- biosenzitivní techniky MeSH
- kardiomyocyty cytologie MeSH
- lidé MeSH
- mikroskopie atomárních sil * přístrojové vybavení metody MeSH
- pluripotentní kmenové buňky cytologie MeSH
- preklinické hodnocení léčiv MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.
- MeSH
- bakteriofágy * MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- povrchová plasmonová rezonance MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Air nanobubbles and nanopancakes were investigated in situ by both tapping mode atomic force microscopy (TM AFM) and atomic force nanolithography techniques employing bovine serum albumin (BSA) film supported by highly oriented pyrolytic graphite (HOPG). The BSA denaturation induced by the water-to-ethanol exchange served for conservation of nanobubble and nanopancake sites appearing as imprints in BSA film left by gaseous cavities formerly present on the interface in the aqueous environment. Once the BSA film was gently removed by the nanoshaving technique applied in ethanol, a clean basal plane HOPG area with well-defined dimensions was regenerated. The subsequent reverse ethanol-to-water exchange led to the re-formation of nanopancakes specifically at the nanoshaved area. Our approach paves the way for the study of gaseous nanostructures with defined dimensions, formed at solid-liquid interface under controlled conditions.
- MeSH
- ethanol MeSH
- grafit chemie MeSH
- mikroskopie atomárních sil MeSH
- nanostruktury chemie ultrastruktura MeSH
- nanotechnologie MeSH
- plyny MeSH
- povrchové vlastnosti MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- voda MeSH
- vzduch MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.
- MeSH
- bakteriální proteiny MeSH
- esterasy MeSH
- exprese genu MeSH
- financování organizované MeSH
- ketony farmakologie chemie MeSH
- lidé MeSH
- ligandy MeSH
- mikroskopie atomárních sil MeSH
- multienzymové komplexy genetika chemie MeSH
- NADH, NADPH oxidoreduktasy genetika chemie MeSH
- nukleocytoplazmatické transportní proteiny genetika chemie MeSH
- rekombinantní fúzní proteiny genetika chemie ultrastruktura MeSH
- silikáty hliníku chemie MeSH
- terciární struktura proteinů MeSH
- Thermus thermophilus enzymologie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
Particles of DeltaProCANC, a fusion of capsid (CA) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particles are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, DeltaProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV DeltaProCANC.
- MeSH
- HIV * ultrastruktura MeSH
- lidé MeSH
- makromolekulární látky MeSH
- Masonův-Pfizerův opičí virus * ultrastruktura MeSH
- mikroskopie atomárních sil MeSH
- sestavení viru MeSH
- virion * ultrastruktura MeSH
- virové proteiny izolace a purifikace metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
