Biomedical subjects

R R Schröder

Publications and source records attributed to R R Schröder.

11 recordsLinked to original sources

PubMed2006

Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate.

Imaging of weak amplitude and phase objects, such as unstained vitrified biological samples, by conventional transmission electron microscopy (TEM) suffers from poor object contrast since the amplitude and phase of the scattered electron wave change only very little. In phase contrast light microscopy the imaging of weak phase objects is greatly enhanced by the use of a quarter-wave phase plate, which produces high signal contrast by shifting the phase of the scattered light. An analogous quarter-wave plate for the electron microscope, designed as an electrostatic einzel lens, was proposed by Boersch in 1947 but the small dimensions of the device have impeded its realization up to now. We here present the first fabrication and application of a miniaturized electrostatic einzel lens driven as TEM quarter-wave phase plate. Phase modulation is generated by the electrostatic field confined to the inside of a microstructured ring electrode. This field affects the phase velocity of the unscattered part of the electron wave. By varying its strength the phase shift of the primary beam can be adjusted to pi/2, producing strong phase contrast independent of spatial frequency. The phase plate proves to be mechanically stable and does not impair image quality, in particular it does not reduce the high-resolution signal. The expected residual lens effect of the einzel lens is minimal. Our microlens is supported by conducting rods arranged in a threefold symmetry. This particular geometry provides optimized single-sideband signal transfer for spatial frequencies otherwise obstructed by the supporting rods.

Journal Article↗

PubMed2004

The structure of the rigor complex and its implications for the power stroke.

Decorated actin provides a model system for studying the strong interaction between actin and myosin. Cryo-energy-filter electron microscopy has recently yielded a 14 A resolution map of rabbit skeletal actin decorated with chicken skeletal S1. The crystal structure of the cross-bridge from skeletal chicken myosin could not be fitted into the three-dimensional electron microscope map without some deformation. However, a newly published structure of the nucleotide-free myosin V cross-bridge, which is apparently already in the strong binding form, can be fitted into the three-dimensional reconstruction without distortion. This supports the notion that nucleotide-free myosin V is an excellent model for strongly bound myosin and allows us to describe the actin-myosin interface. In myosin V the switch 2 element is closed although the lever arm is down (post-power stroke). Therefore, it appears likely that switch 2 does not open very much during the power stroke. The myosin V structure also differs from the chicken skeletal myosin structure in the nucleotide-binding site and the degree of bending of the backbone beta-sheet. These suggest a mechanism for the control of the power stroke by strong actin binding.

Actins↗

PubMed2000

New high-DQE imaging plate scanner using the reflected readout laser signal for noise corrections.

Imaging Plates (IPs) are in principle ideal electron detectors combining a large active layer area with a high sensitivity, linear dynamic range detection over 5 orders of magnitude. A moderate resolution and a decreasing detection quantum efficiency (DQE) for higher electron doses limit their use so far. The decrease of the DQE results from linear noise contributed by readout laser instabilities and inhomogeneities of the IP active layer. Here we present data on a new IP drum scanner prototype. This scanner combines twin channel amplification electronics with a new type of readout laser which allows a smaller readout focus and increased stability. The current nominal pixel size is 25 microm, and the measured modulation transfer function (MTF) indicates that further reduction of the scanning step size down to pixel sizes in the range of 12-15 microm should be possible. A unique feature of the new scanner is the simultaneous recording of the reflected readout laser light. The reflected light signal can be used for a posteriori alignment of repeated scans of one individual IP and for a correction of one part of the high spatial frequency noise contribution (reflected light correction). The posteriori alignment now allows an easy conventional gain normalization of the luminescence signal without using special markers on the IP. Both corrections lead to an increase of the DQE for high electron doses.

Biophysical Phenomena↗

PubMed2000

Zero-loss image formation and modified contrast transfer theory in EFTEM.

For a weak phase/weak amplitude object the information transfer in the imaging process of TEM is described by the common formalism of the contrast transfer function (CTF). So far the effects of inelastic scattering were not accounted for in this formalism. In conventional imaging they were simply neglected. In energy filtering TEM (EFTEM), where removal of inelastic electrons leads to higher specimen contrast, they were modelled by a global increase of the elastic amplitude contrast. Thus, the description of inelastic and elastic scattering was mixed. Here a new ansatz is proposed which treats elastic and inelastic contrast transfer separately by adding an inelastic contribution to the scattering potentials. In EFTEM this has the effect of adding a filter contrast which depends on the characteristics of the inelastic scattering. For samples with dominant plasmon loss the additional filter contrast is restricted to low resolution. Because of its strong dependence on the nature of the inelastic scattering process, the filter contrast cannot in general be unified with the conventional elastic amplitude contrast. The modified CTF theory for EFTEM was tested experimentally on a variety of samples. Images of amorphous layers of copper, aluminium, and carbon films, as well as zero-loss images of proteins embedded in amorphous ice were evaluated. The values of the parameters of the additional filter contrast were determined for carbon film and proteins embedded in vitrified ice. Comparison of different CTF models used to reconstruct 3D volumes from zero-loss images confirmed that best agreement with the atomic model is attained with the new, modified CTF theory.

Actins↗

PubMed1999

Quantification of total calcium in terminal cisternae of skinned muscle fibers by imaging electron energy-loss spectroscopy.

Skinned muscle fibers are ideal model preparations for the investigation of Ca2+ -regulatory mechanisms. Their internal ionic milieu can be easily controlled and distinct physiological states are well defined. We have measured the total Ca content in the terminal cisternae of such preparations using imaging electron energy-loss spectroscopy (Image-EELS) as a new approach for quantification of sub-cellular element distributions. Murine muscle fibers submitted to a standardized calcium-loading procedure were cryo-fixed with a combined solution exchanger/plunge freezing device. Energy-filtered image series were recorded from ultrathin freeze-dried cryosections of samples immobilized in either relaxed or caffeine-contracted state. From these image series, electron energy-loss spectra were extracted by digital image-processing and quantitatively processed by multiple-least-squares-fitting with reference spectra. The calculated fit coefficients were converted to Ca-concentrations by a calibration obtained from Ca-standards. Total Ca-contents in the terminal cisternae of skinned skeletal muscle fibers decreased upon caffeine-induced Ca-release from 123+/-159 (+/-11) to 73+/-102 (+/-8) mmol/kg d.w. (weighted mean +/- SD (+/-SEM)).

Animals↗

PubMed1993

Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1.

Elucidation of the molecular contacts between actin and myosin is central to understanding the force-generating process in muscle and other cells. Actin, a highly conserved globular protein found in all eukaryotes, polymerizes into filaments (F-actin) for most of its biological functions. Myosins, which are more diverse in sequence, share a conserved globular head of about 900 amino acids in length (subfragment-1 or S1) at the N-terminal end of the molecule. S1 contains all the elements necessary for mechano-chemical force transduction in vitro. Here we report an atomic model for the actomyosin complex produced by combining the atomic X-ray structure of F-actin and chicken myosin S1 with a three-dimensional reconstruction from electron micrographs of frozen-hydrated F-actin decorated with recombinant Dictyostelium myosin S1. The accuracy of the reconstruction shows the position of actin and myosin molecules unambiguously.

Actins↗

PubMed1992

Cryo-electron microscopy of vitrified muscle samples.

A great deal of information on the 3-dimensional structure of the protein assemblies involved in muscle contraction has been obtained using conventional transmission electron microscopy. In recent years, developments in cryo-electron microscopy have facilitated work with fully hydrated, non-chemically fixed specimens. It is shown how this technique can be used to visualize muscle sarcomere filaments in quasi-native conditions, to access hitherto inaccessible states of the crossbridge cycle, and to obtain new high resolution structural information on their 3-dimensional protein structure. A short introduction to the crossbridge cycle and its biochemically accessible states illustrates the problems amenable to studies using the electron microscope, as well as the possibilities offered by cryo-microscopy on vitrified samples. Work on vitrified cryo-sections and myosin filament suspensions demonstrates the accessibility of crossbridge states and gives implications on the gross structural features of myosin filaments. Recent studies on actin filaments and myosin (S1) decorated actin filaments provide the first high resolution data on vitrified samples. The use of photolabile nucleotide precursors allows the trapping of short lived states in the millisecond time range, thereby visualizing intermediate states of the crossbridge cycle.

Animals↗

PubMed1992

Zero-loss energy-filtered imaging of frozen-hydrated proteins: model calculations and implications for future developments.

Energy-filtered transmission electron microscopes operating in zero-loss mode are used increasingly to study biological material in frozen-hydrated conditions. The contrast enhancement and improved structural resolution obtainable by this method have been studied using Monte-Carlo model calculations for the scattering processes occurring in such samples. Three models representing typical situations have been analysed, each normalized to minimal beam damage. It is shown that for proteins in thin layers of ice an optimal signal-to-noise ratio is achieved in the 80-120-keV electron energy range. For proteins which have to be embedded in thicker ice layers, a considerably higher acceleration voltage is required. In particular, electron energies above 200 keV would be desirable for electron diffraction work on microcrystals.

Animals↗

PubMed1991

Time-resolved cryo-electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides.

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.

Actins↗

PubMed1990

Cryo-electron microscopic studies of relaxed striated muscle thick filaments.

Electron micrograph images of rapidly frozen suspensions of thick filaments from four different muscle types are presented. Their optical and computer transforms are compared with images and diffraction patterns of negatively stained filaments and with X-ray data from the same muscles. We conclude that myosin head arrangement can be preserved on rapid freezing and that the images produced can be analysed by image processing techniques to give new information on thick filament structure.

Actin Cytoskeleton↗

PubMedDate not supplied

Polymerizing properties of pepstatin A.

Pepstatin A, a pentapeptide aspartyl protease inhibitor, can spontaneously polymerize into filaments having a helical substructure and, after negative staining, characteristic diameters ranging from 6 to 12 nm. Optical diffraction analysis demonstrated that these filaments consist of a 6-nm-wide strand helically wound with a periodic pitch of 25 nm. Selected images suggest that these filaments may actually be composed of two, intertwined 6-nm-wide strands, an hypothesis not at variance with the diffraction data. These filaments may extend over several micrometers. At low ionic strength and neutral pH, the critical concentration for pepstatin A filament assembly is 0.1 mM. At higher pepstatin A concentrations or in physiological salt solutions, a variety of higher order structures were observed, including ribbons, sheets, and cylinders with both regular and twisted or irregular geometries. Pepstatin A polymerized into these higher order structures loses its ability to inhibit the aspartyl protease of the human immunodeficiency virus type 1. These results have implications not only for model studies on the polymerization of small peptides into higher order structures, but also for the practical development of soluble protease inhibitors.

Microscopy, Electron↗