662 research outputs found
Paving the way for structural modelling by smFRET measurements
No full textThe thesis at hand contributes to the field of structure determination of bio-macromolecules. It focuses on the Förster resonance energy transfer (FRET), which allows to infer distances at the molecular level (≈ 2-10 nm) making FRET a promising tool for structure analysis. In the process of FRET, the energy of a photon, absorbed by a fluorophore, called donor, is transferred to a second dye, called acceptor. FRET experiments result in an average FRET efficiency which in the first place depends on the distance between donor and acceptor. If the two dyes are attached to specific sites of a macromolecule, we can infer intramolecular distances not only in vitro, but even in vivo. Furthermore, FRET can be observed on a single-molecule level in real time. Thus, different conformations and their dynamics of one macromolecule are observable and distinguishable by single-molecule FRET (smFRET) experiments.
These features encouraged the development of the Nano-Positioning System (NPS). The basic idea of NPS is that we can localise an unknown position unambiguously, if we know its distances to at least four known positions. Given a network of dyes, NPS allows thus for the dependent localisation of unknown positions of fluorophores, such that the structure of macromolecular complexes can be illuminated by FRET measurements. As a Bayesian analysis tool, the theoretic basis of NPS is a probability distribution, the so-called posterior. It tells us how probable a spatial arrangement of dyes in the network is conditional on the experimental smFRET efficiencies measured between the dyes. Hence, the present thesis deals on the one hand with the development and implementation of a fast and adaptive sampling algorithm which extracts the structural information hidden within the posterior leading to the release of Fast-NPS. On the other hand, we promoted smFRET as a structure analysis method by providing a manual guide of how to perform smFRET measurements at a TIRF microscope and how to subsequently infer a structure in using the software Fast-NPS. This is not only presented in a manuscript, but also in a how-to video.
A major difficulty in the inference of structures by smFRET measurements is the transformation of the smFRET efficiency to a distance. An important factor here is how the fluorophores are described. The publications of the cumulative thesis at hand report the progressing development of dye models from one single conservative model to a set of primitive models incorporating different assumptions. For the application of the latter, we developed a consistency test such that we can test, if the experimental smFRET efficiency is still in accordance with the applied dye models. This sophisticated procedure led to a 4-fold enhancement of localization precision on average.
In a benchmark study, we applied these models to analyse smFRET data obtained from dsDNA and a protein-DNA complex at a TIRF microscope. We could show that Fast-NPS infers the correct position in both cases. However, we saw that some models are too strict in their assumptions, such that the inferred structures become inconsistent with the smFRET data. A further concern is that we could also find model combinations that do not infer the correct positions, but still are consistent with the experimental data.
The latter problem guided us to a more sophisticated theory of dye models. We understood that the FRET efficiency is inseparably determined by the geometry and kinetics of both dyes. This leads to a complete description of the smFRET efficiencies by expectation values. For dyes whose motions can be treated approximately as time-independent, we can compute the expected value of the smFRET efficiency by Monte Carlo integration. Completing the theory, we also established a stochastic simulation in order to calculate the expected value in the case of time-dependent motions. We accomplished this by explicitly simulating the rotational and translational diffusion of donor and acceptor simultaneously to the donor de-excitation. It is important to note that these simulations are guided by experimental parameters obtained from fluorescence lifetime and time-resolved anisotropy measurements.
In further utilizing the stochastic simulation, we developed a statistical method to classify the motions of donor and acceptor during a single de-excitation event into one of the so-called transfer regimes telling us how to average the motion. Applying this method, an investigation of dyes attached to dsDNA showed us that their kinetic assumptions, usually applied in structural inference, are not valid over the whole distance range inferred by FRET. This has a great impact on the future analysis of smFRET experiments
High-field diffusion tensor imaging of mouse brain in vivo using single-shot STEAM MRI
No full textInformation about the microstructural organization of cerebral white matter that is accessible by magnetic resonance diffusion tensor imaging (DTI) gains increasing importance for studies of animal brain. Particular challenges occur for in vivo conditions as well as at high magnetic fields. Here, we have employed a diffusion-weighted (DW) single-shot STEAM MRI sequence for DTI of mouse brain in vivo at 7 T. The approach exploits the increased longitudinal magnetization and prolonged T1 relaxation times of water protons at higher magnetic field strengths without suffering from susceptibility-induced artifacts. When compared to studies at 2.35 T, half Fourier DW STEAM MRI at 7 T yielded a substantial gain in signal-to-noise ratio (SNR) that could be invested either in a reduction of the measurement time or an increase of the spatial resolution. Thus, for a measurement time of 3h, DTI with a voxel size of 117 microm x 117 microm x 720 microm not only resulted in high-quality maps of the fractional anisotropy and main diffusion direction (MDD), but also allowed for fiber tracking of major mouse brain structures in vivo
Chromium(VI) as a novel MRI contrast agent for cerebral white matter: Preliminary results in mouse brain in vivo
No full textThis work demonstrates that intraventricular microinjections of a low dose of potassium dichromate (0.4 μL of 10 mM solution) yield a specific contrast enhancement of white matter (WM) tracts in T1-weighted 3D MRI of mouse brain in vivo. Pronounced and persistent signal increases (40–100% at 24 hr after injection) were observed in the corpus callosum, anterior commissure, fornix, and stria medullaris, as well as in the mammillothalamic tract and fasciculus retroflexus. These results suggest that the extracellular diffusion of diamagnetic chromium(VI) (Cr(VI)) after injection is followed by a tissue-specific reduction to paramagnetic Cr(V) and (III), which relies predominantly on the oxidation of myelin lipids. Because Cr(VI)-induced contrast leads to only a mild unspecific enhancement (10–20%) of gray matter (GM) structures, such as the hippocampal formation, the method reveals novel information that differs from that obtainable using other paramagnetic ions, such as manganese
In vivo 3D MRI staining of mouse brain after subcutaneous application of MnCl2
No full textFollow-up T1-weighted 3D gradient-echo MRI (2.35 T) of murine brain in vivo (N = 5) at 120 μm isotropic resolution revealed spatially distinct signal increases 6–48 hr after subcutaneous application of MnCl2 (20 mg/kg). The effects result from a shortening of the water proton T1 relaxation time due to the presence of unchelated paramagnetic Mn2+ ions, which access the brain by systemic circulation and crossing of the blood–brain barrier (BBB). A pronounced Mn2+-induced signal enhancement was first seen in structures without a BBB, such as the choroid plexus, pituitary gland, and pineal gland. Within 24 hr after administration, Mn2+ contrast highlighted the olfactory bulb, inferior colliculi, cerebellum, and the CA3 subfield of the hippocampus. The affinity of Mn2+ to various brain systems suggests the neuronal uptake of Mn2+ ions from the extracellular space and subsequent axonal transport. Thus, at least part of the Mn2+ contrast reflects a functional brain response of behaving animals, for example, in the olfactory system. In vivo MRI staining of the brain by systemic administration of MnCl2 may contribute to phenotyping mutant mice with morphologic and functional alterations of the central nervous system. Magn Reson Med 48:852–859, 2002. © 2002 Wiley-Liss, Inc
Observations on Strict Derivational Minimalism
No full textMichaelis J. Observations on Strict Derivational Minimalism. Electronic Notes in Theoretical Computer Science. 2004;53:192-209
Mapping of the habenulo-interpeduncular pathway in living mice using manganese-enhanced 3D MRI
No full textThis magnetic resonance imaging (MRI) study describes mapping of the habenulo-interpeduncular pathway in living mice based on manganese-induced contrast. Six hours after intracerebroventricular microinjection of MnCl2, T1-weighted 3D MRI (2.35 T) at 117 μm isotropic resolution revealed a continuous pattern of anterograde labeling from the habenula via the fasciculus retroflexus to the interpeduncular nucleus. Alternatively, the less invasive systemic administration of MnCl2 allowed for monitoring of the dynamic uptake pattern of respective neural components with even higher reproducibility across animals. Time courses covered the range from 42 min to 24 h after injection. In conclusion, manganese-enhanced MRI may open new ways for functional assessments of the habenulo-interpeduncular system in animal models with cognitive impairment
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No full textMagnetization transfer MRI of mouse brain reveals areas of high neural density
No full textExtending applications of magnetization transfer contrast (MTC) in magnetic resonance imaging (MRI) of the human central nervous system, this work quantitatively describes MTC of the murine brain. As a novel finding, complementing T1- and T2-weighted MRI, MTC allows for the distinction of densely packed gray matter from normal gray and white matter. Examples include the Purkinje cell layer and the granular cell layer in the mouse cerebellum as well as the delineation of the CA3 subfield of the hippocampus relative to surrounding hippocampal gray matter and white matter tracts such as the hippocampal fimbria. Using a kainate lesion model, the CA3 hyperintensities in MTC and T1-weighted MRI are assigned to the densely packed somata of pyramidal cells
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No full textHalogenated volatile anesthetics alter brain metabolism as revealed by proton magnetic resonance spectroscopy of mice in vivo
No full textHalogenated volatile anesthetics (HVA) are widely used in medicine and research but their effects on brain metabolism in intact organisms are still largely unknown. Here, localized proton magnetic resonance spectroscopy (MRS) of anesthetized mice was applied to evaluate HVA effects on cerebral metabolites in vivo. Experimental protocols combined different concentrations of isoflurane, halothane, sevoflurane, and desflurane with known modulators of adrenergic, GABAergic, and glutamatergic neurotransmission. As a most striking finding, brain lactate increased in individual mice from 1.0 ± 0.6 mM (awake state) to 6.2 ± 1.5 mM (1.75% isoflurane). In addition, relative to total creatine, there were significant isoflurane-induced increases of alanine by 111%, GABA by 20%, choline-containing compounds by 20%, and myo-inositol by 10% which were accompanied by significant decreases of glucose by 51% and phosphocreatine by 9%. The elevation of lactate was most pronounced in the striatum. The HVA effects correlated with the respective minimal alveolar concentrations and were mostly reversible within minutes. The observed alterations are best explained by an HVA-induced stimulation of adrenergic pathways in conjunction with an inhibition of the respiratory chain. Apart from casting new light on cerebral energy metabolism, the present results challenge brain studies of HVA-anesthetized animals
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