Optimized and far-red-emitting variants of fluorescent protein eqFP611

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.     

Shear induced relaxation of polymer micelles at the solid-liquid interface

A 20% aqueous solution of (ethylene oxide) 99-(propylene oxide) 65-(ethylene oxide) 99, F127, was investigated by combining rheology in a cone/plate-geometry and surface-sensitive grazing incident neutron scattering. The crystalline structure formed by the polymer micelles becomes less pronounced for low shear rates, but correlations increase for higher shear rates. After stopping shear a slow relaxation of the micelles is found in the vicinity (50 mum thick layer) of a hydrophilic silicon wall (strong micelle-wall interaction), while a fast relaxation is observed in the boundary layer against the hydrophobic silicon wall (weak micelle-wall interaction). The results show that in the vicinity of the interface wall-particle interactions compete heavily with the shear force acting on the liquid.     

Gated trapped ion mobility spectrometry coupled to fourier transform ion cyclotron resonance mass spectrometry

Analysis of molecules by ion mobility spectrometry coupled with mass spectrometry (IMS-MS) provides chemical information on the three dimensional structure and mass of the molecules. The coupling of ion mobility to trapping mass spectrometers has historically been challenging due to the large differences in analysis time between the two devices. In this paper we present a modification of the trapped ion mobility (TIMS) analysis scheme termed “Gated TIMS” that allows efficient coupling to a Fourier Transform Ion Cyclotron Resonance (FT-ICR) analyzer. Analyses of standard compounds and the influence of source conditions on the TIMS distributions produced by ion mobility spectra of labile ubiquitin protein ions are presented. Ion mobility resolving powers up to 100 are observed. Measured collisional cross sections of ubiquitin ions are in excellent qualitative and quantitative agreement to previous measurements. Gated TIMS FT-ICR produces results comparable to those acquired using TIMS/time-of-flight MS instrument platforms as well as numerous drift tube IMS-MS studies published in the literature.     

Conformational preferences of chondroitin sulfate oligomers using partially oriented NMR spectroscopy of 13C-labeled acetyl groups

A new method is presented for the retrieval of information on the conformation of glycosaminoglycan oligomers in solution. The method relies on the replacement of acetyl groups in isolated native oligomers with 13C labeled acetyl groups and the extraction of orientational constraints from residual dipolar couplings (RDCs) and chemical shift anisotropy (CSA) offsets observed in NMR spectra of partially oriented samples. A novel method for assignment of resonances based on the correlation of resonance intensities with isotope ratios determined from mass spectrometric analysis is also presented. The combined methods are used in conjunction with more traditional NMR structural data to determine the solution structure of a pentasaccharide, GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S(beta1-4)GlcA(beta1-3)GalNAc4S-ol, derived by enzymatic hydrolysis of chondroitin sulfate. The geometry derived is compared to that for similar molecules that have been reported in the literature, and prospects for use of the new types of data in the study of protein-bound oligosaccharides are discussed.     

A first-principles study of the electronic and structural properties of gamma-TaON

The geometric and electronic structure of the recently found new polymorph of tantalum oxynitride, gamma-TaON, and its structural stability were studied quantum-chemically at the density functional level. Results obtained by complementary quantum-chemical techniques with wavefunctions either expanded in atom-centered functions or in plane waves were compared, having employed pure density-functional functionals within the generalized gradient approximation as well as density-functional/Hartree-Fock hybrid methods. In particular, several plausible anion distributions were investigated and, in accordance with Pauling's second rule, it was found that the configuration in which nitrogen occupies crystallographic sites with highest coordination numbers is the most stable one. Theoretically generated local structural parameters were used to improve the accuracy of the experimentally derived information. The bonding situation in the most stable configuration was investigated by an analysis of the density of states.     

Investigation into the factors affecting accuracy of mass measurements on a time-of-flight mass spectrometer using Design of Experiment

The results of an investigation of the parameters which have the most significant effect on the accuracy of mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (q-oaToF) are reported. The influence of eight factors is investigated: ion abundances of reference and analyte compounds, mass difference between analyte and reference compounds, quality of calibration, number of reference acquisitions averaged and TDC (time-to-digital converter) settings (resolution, Np multiplier (number of pushes correction factor), minimum number of points, i.e. minimum acquisition width which defines a peak). To extract the maximum information from as few experiments as possible, a Design of Experiment approach was used. The data will be used as a basis for developing guidance on accurate mass measurement on q-oaToF instruments.     

Simultaneous temperature and 2D velocity measurements in a turbulent heated jet using combined laser-induced fluorescence and LDA

 This paper presents an efficient technique for the characterization of thermal transport properties in turbulent flows. The method is based on the temperature dependence of fluorescence, induced by laser radiation, of an organic dye. The laser-induced fluorescence technique is combined with 2D laser Doppler anemometry, in order to measure in the same sample volume simultaneously and instantaneously the temperature and velocity. The technique is demonstrated on a turbulent heated round jet: the mean and fluctuating dynamic and thermal fields are investigated, and the temperature-velocity cross-correlations are determined in order to characterize the turbulent diffusivity and the turbulent Prandtl number. Received: 30 October 1997/Accepted: 14 July 1998     

Mathematik als Grundlage beruflicher Kompetenz

A concept of teaching basic mathematical knowledge and skills related to the vocational training of master craftsmen in the field of electrical engineering and metal engineering is described. The provision of application-oriented mathematics is pointed out as an important basis of competence needed by master craftsmen in their professional surroundings. A framework for the development of training supporting materials for mathematics learning is explained on the basis of earlier experiences as well as on newer constructivitically oriented instructional approaches of educational psychology and in consideration of current professional guidelines and examination regulations of the Chambers of Industry and Commerce. These materials are developed into three CBT-units which are to be of fundamentally different design in order to enable learning on different levels using the material on the learners' own initiative. Active self-organization is offered to the course participants via the use of interactive multimedia-based presentations.     

Speckle correlation for the analysis of random processes at rough surfaces

The importance of technological processes like corrosion, ablation or deposition causes interest in the quantitative monitoring of changes at rough surfaces. Thus, there is a need for effective methods to measure the statistical parameters characterizing changes in the profile or the material composition of such objects. The speckle field scattered from the surface is used as information carrier and its change is measured by correlation. This is realized by sophisticated data acquisition and digital processing techniques. An important issue is the interpretation of the correlation output in terms of statistical parameters describing the surface change. For many random surfaces a geometrical relation between surface profile and optical phase proves satisfactory. This allows to determine the standard deviation of the profile change. For a verification, speckle decorrelation in model surfaces of known deviation is measured. The paper introduces the speckle correlation concept, outlines some history and current setups and describes methods for data evaluation. The reliability of the quantitative interpretation of the speckle decorrelation is demonstrated. The method is illustrated by studies of metal corrosion and material removal in the cleaning of historical objects by laser ablation.