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Eingeladener Vortrag
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Die vorgelegte Arbeit zeigt die Ausarbeitung und die Überprüfung einer Theorie, mit welcher die Ergebnisse aus einem neu entwickelten Mess-Verfahren (DySEM-Technik) beschrieben werden können. Mit dem Begriff “DySEM“ (Dynamic Scanning Electron Microscopy) wird ein experimentelles Verfahren bezeichnet, bei dem ein Elektronenstrahl als Mess-Sonde über einem mikroskaligen Schwinger verfahren wird, wobei die Schwingung durch eingesetzte Lock-In Technik frequenzaufgelöst dargestellt werden kann. Neben dem klassischen Sekundärelektronen-Signal wird zur Bildgebung auch der Anteil aus dem Signal genutzt, der sich anregungssynchron ändert. Die DySEM-Technik ermöglicht eine direkte Visualisierung der Schwingungsdynamik der oszillierenden Struktur, da zwischen unterschiedlichen Eigenmoden (flexural, torsional) als auch den jeweiligen höheren Harmonischen optisch eindeutig unterschieden werden kann. Damit bietet sich dieses Verfahren als ein Werkzeug der Modal-Analyse mikroskaliger Schwinger an, welche in mikro- bzw. nanoelektromechanischen Systemen (MEMS bzw. NEMS) häufig Verwendung finden und bei denen eine Optimierung der Designparameter oft erst durch die Bildgebung der Schwingung zu erreichen ist. Zusätzlich zeigen die DySEM-Bilder charakteristische Amplituden-abhängige Bildmerkmale, die theoretisch verstanden werden müssen. Prinzipiell ist die DySEM-Technik nicht an den Elektronenstrahl als Mess-Sonde gekoppelt. Allerdings erweist sich gerade im Zuge fortschreitender Miniaturisierung mit immer kleinskaligeren Schwingern eine elektronenoptische Orts-Auflösung als günstig. Bei der theoretischen Analyse des Abbildungsmechanismus liegt der Fokus auf der Untersuchung der raum-zeitlichen Dynamik der Wechselwirkung zwischen Elektronenstrahl und der periodisch darunter hinweg schwingenden Mikrostruktur, für die erstmals ein umfängliches Modell abgeleitet werden konnte, wodurch die detaillierte Interpretation der experimentellen Ergebnisse möglich wurde. Zusätzlich spielen lokale Eigenschaften (Materialeigenschaften) des Schwingers eine Rolle. Ebenso müssen die Beiträge von Energieverlustmechanismen zur Bildgebung berücksichtigt werden. Um die bildgebenden Gleichungen explizit ableiten zu können, beschränkt sich die mathematische Analyse in dieser Arbeit auf die Annahme eines frei oszillierenden, einseitig geklemmten Schwingers ohne Wechselwirkung mit Materie, wie es im DySEM-Experiment durch die Bildgebung im Hochvakuum angenähert wird. Die aufgrund dieses Modells simulierten DySEM-Bilder stimmen mit den experimentell gewonnenen Ergebnissen qualitativ und quantitativ gut überein.
Theoretical analysis of vibrating microstructuresusing Dynamic Scanning Electron Microscopy (DySEM)
(2015)
We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSB) and double-strand breaks (DSB), was determined by electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of DSB to SSB was found to be (1:12) as compared to 1:88) found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, cosolutes) for an electron energy range which is difficult to probe by standard methods.
Structural changes in plasmid DNA caused by radiation and its protection by Ectoine: an AFM analysis
(2017)
Most ionizing radiation in water ends in an avalanche of low energy electrons which play a dominant role together with OH-radicals in damaging DNA. In the present study we irradiated plasmid DNA with electrons (primary energy 30keV) under physiological conditions, performed with as well as without Ectoine. Ectoine is a compatible solute, synthesized and accumulated in molar concentration within bacteria to withstand osmotic stress or different other stressors.
Plasmid DNA (pUC19, 2686 bp) was studied due to its supercoiled isoform which is highly sensitive to radiation damage. In biochemistry gel electrophoresis is applied for structural analysis of DNA. Although it is a standard technique, a reliable discrimination of short fragments caused by radiation is often difficult. AFM is also commonly used for imaging susceptible biomolecules and, since it is based on a single molecule observation, for analysis of contour lengths of linear DNA as well. Therefore, in our study the structural changes in plasmid DNA after irradiation with different doses were quantitatively analyzed by means of intermittent contact AFM. The figure shows representative AFM images of electron irradiated pUC19 DNA (bar=200nm). For AFM imaging the DNA was chemically fixed on ultra-smooth mica. As can be clearly seen, with increasing radiation dose the number of undamaged DNA declines and fragmented DNA arises (A, B). In aqueous Ectoine solution (1M) the effect of radiation on DNA is dramatically depressed. Ectoine apparently confers protection even against high radiation: the plasmids remain predominantly in the supercoiled isoform (D). Therefore, we strongly believe that Ectoine is a potent protective substance of DNA against ionizing radiation.
Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the use of ectoine as an ˙OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).
Ectoine plays an important role in protecting biomolecules and entire cells against environmental stressors such as salinity, freezing, drying and high temperatures. Recent studies revealed that ectoine also provides effective protection for human skin cells from damage caused by UV-A radiation. These protective properties make ectoine a valuable compound and it is applied as an active ingredient in numerous pharmaceutical devices and cosmetics. Interestingly, the underlying mechanism resulting in protecting cells from radiation is not yet fully understood. Here we present a study on ectoine and its protective influence on DNA during electron irradiation. Applying gel electrophoresis and atomic force microscopy, we demonstrate for the first time that ectoine prevents DNA strand breaks caused by ionizing electron radiation. The results presented here point to future applications of ectoine for instance in cancer radiation therapy.
We use a dynamic scanning electron microscope (DySEM) to analyze the movement of oscillating micromechanical structures. A dynamic secondary electron (SE) signal is recorded and correlated to the oscillatory excitation of scanning force microscope (SFM) cantilever by means of lock-in amplifiers. We show, how the relative phase of the oscillations modulate the resulting real part and phase pictures of the DySEM mapping. This can be used to obtain information about the underlying oscillatory dynamics. We apply the theory to the case of a cantilever in oscillation, driven at different flexural and torsional resonance modes. This is an extension of a recent work (Schr¨oter et al 2012 Nanotechnology 23 435501), where we
reported on a general methodology to distinguish nonlinear features caused by the Imaging process from those caused by cantilever motion.
We use a dynamic scanning electron microscope (DySEM) to map the spatial distribution of the vibration of a cantilever beam. The DySEM measurements are based on variations of the local secondary electron signal within the imaging electron beam diameter during an oscillation period of the cantilever. For this reason, the surface of a cantilever without topography or material variation does not allow any conclusions about the spatial distribution of vibration due to a lack of dynamic contrast. In order to overcome this limitation, artificial structures were added at defined positions on the cantilever surface using focused ion beam lithography patterning. The DySEM signal of such high-contrast structures is strongly improved, hence information about the surface vibration becomes accessible. Simulations of images of the vibrating cantilever have also been performed. The results of the simulation are in good agreement with the experimental images.
A specially designed scanning force microscope (SFM, atomic force microscope – AFM) was incorporated into the chamber of a commercial scanning electron microscope (SEM) to investigate vibrating SFM cantilevers at their resonance. Inherently, the spatial resolution of electron microscopy is higher than that of optical methods. In this paper we present vibration modes (eigenmodes) of two different SFM cantilevers. Their nonlinear behavior is also explored in order to depict their 2nd harmonics (twice the fundamental frequency). Imaging of the local vibration is performed by measuring the frequency- and phase-selective responses of the SE signal at different X and Y positions of the scanned electron beam.
Analytical and numerical analysis of imaging mechanism of dynamic scanning electron microscopy
(2012)
The direct observation of small oscillating structures with the help of a scanning electron beam is a new approach to study the vibrational dynamics of cantilevers and microelectromechanical systems. In the scanning electron microscope, the conventional signal of secondary electrons (SE, dc part) is separated from the signal response of the SE detector, which is correlated to the respective excitation frequency for vibration by means of a lock-in amplifier. The dynamic response is separated either into images of amplitude and phase shift or into real and imaginary parts. Spatial resolution is limited to the diameter of the electron beam. The sensitivity limit to vibrational motion is estimated to be sub-nanometer for high integration times. Due to complex imaging mechanisms, a theoretical model was developed for the interpretation of the obtained measurements, relating cantilever shapes to interaction processes consisting of incident electron beam, electron–lever interaction, emitted electrons and detector response. Conclusions drawn from this new model are compared with numerical results based on the Euler–Bernoulli equation.
Strand breaks and conformational changes of DNA have consequences for the physiological role of DNA. The natural protecting molecule ectoine is beneficial to entire bacterial cells and biomolecules such as proteins by mitigating detrimental effects of environmental stresses. It was postulated that ectoine-like molecules bind to negatively charged spheres that mimic DNA surfaces. We investigated the effect of ectoine on DNA and whether ectoine is able to protect DNA from damages caused by ultraviolet radiation (UV-A). In order to determine different isoforms of DNA, agarose gel electrophoresis and atomic force microscopy experiments were carried out with plasmid pUC19 DNA. Our quantitative results revealed that a prolonged incubation of DNA with ectoine leads to an increase in transitions from supercoiled (undamaged) to open circular (single-strand break) conformation at pH 6.6. The effect is pH dependent and no significant changes were observed at physiological pH of 7.5. After UV-A irradiation in ectoine solution, changes in DNA conformation were even more pronounced and this effect was pH dependent. We hypothesize that ectoine is attracted to the negatively charge surface of DNA at lower pH and therefore fails to act as a stabilizing agent for DNA in our in vitro experiments.
In dosimetry the determination of the effectiveness of the damaging processes is standardized and accounted for by the radiation and tissue weighting factor. For the underlying constituents of the tissue, that is the various biomolecules, such a systematic approach doesn't exist. This makes it difficult to compare results obtained under different experimental conditions. In the following work, we will describe a method to obtain comparable values for the radiation-biomolecule interaction, measured under different conditions. This approach can lead to standardization of dosedamage relationship at the molecular level. Such approach is necessary for a better understanding of the relations between the damage of the single constituents of biological tissue and the whole – finally gaining a more complete picture of irradiation damage.
Tapping mode AFM (TM-AFM) is a standard technique to image biomolecules and cells avoiding damage to susceptible samples.
For preparing AFM samples we fixed (untreated and radiated) plasmids chemically on ultra-smooth mica silanized with APTES.
The recorded AFM images were examined concerning the contour length, the conformation and the writhing number of the plasmids.
We found that the measured contour length is in accordance with the number of base pairs. Apart from that, we indicate different structures of plasmids from our AFM images, which were assigned to our plasmid data from agarose gel-electrophoresis.