The grid sectioning technique: a study of catalase platelets.

The grid sectioning technique has been used to obtain the two missing principal axis projections of orthorhombic catalase platelets and to measure directly the unit cell c-value. The negatively stained platelets have a unit cell c-dimension of half that proposed by Unwin (1975) from powder X-ray diffraction. The precision of the grid sectioning technique in positioning sections along a specimen axis shows that the growth fault lines usually observed on negatively stained catalase platelets are rows of missing molecules filled with stain. From these sections conclusions are drawn concerning the action of negative stain on a specimen, the microtomy process, and the specimen/supporting film interaction. Finally the value of microtomy for detailed structural analysis of biological objects is emphasized.     

Removing high contrast artifacts via digital inpainting in cryo-electron tomography: an application of compressed sensing

To cope with poor quality in cryo-electron tomography images, electron-dense markers, such as colloidal goldbeads, are often used to assist image registration and analysis algorithms. However, these markers can create artifacts that occlude a specimen due to their high contrast, which can also cause failure of some image processing algorithms. One way of reducing these artifacts is to replace high contrast objects with pixel densities that blend into the surroundings in the projection domain before volume reconstruction. In this paper, we propose digital inpainting via compressed sensing (CS) as a new method to achieve this goal. We show that cryo-ET projections are sparse in the discrete cosine transform (DCT) domain, and, by finding the sparsest DCT domain decompositions given uncorrupted pixels, we can fill in the missing pixel values that are occluded by high contrast objects without discontinuities. Our method reduces visual artifacts both in projections and in tomograms better than conventional algorithms, such as polynomial interpolation and random noise inpainting.     

A comparison of negatively stained electron micrographs and projections obtained from single crystal X-ray studies: The lipovitellin complex from lamprey

The analysis of single crystals of the lipovitellin complex from lamprey has made it possible to compare electron density projections derived from X-ray diffraction and density modification methods with previously published electron micrographs. The close correlation between the images obtained by the two methods demonstrates that the fidelity of images obtained by electron microscopy is excellent despite the loss of specimen order. These correlations also attest to the ability of density modification methods, currently used in macromolecular crystallography, to estimate the phases of small angle X-ray reflections.     

Development of the multiple sequence approximation within the AGADIR model of α-helix formation: Comparison with Zimm-Bragg and Lifson-Roig formalisms

In this work we present the development of the multiple sequence approximation (AGADIRms) and the standard one-sequence approximation (AGADIRIs) within the framework of AGADIR's α-helix formation model. The extensive comparison between these new formulations and the original one [AGADIR; v. Muñoz and L. Serrano (1994),Nat. Struct. Biol., Vol. 1, pp. 399–409] indicates that the standard one-sequence approximation is virtually identical to the multiple sequence approximation, while the previously used residue partition function approximation [Muñoz and Serrano (1994); (1995), J. Mol. Biol., Vol. 245, pp. 275–296] is less precise. The calculations of the average helical content performed with AGADIR are precise for peptides of less than 30 residues and progressively diverge from the multiple sequence formulation for longer peptides. The helicity distribution of heteropolypeptides with less than 50% average helical content is also well described, while those of quasi-homopolymers with high helical content tend to be flattened. These inaccuracies lead to an underestimation of 0.017 kcal/mol for the mean-residue enthalpic contribution in AGADIR, as compared to AGADIRms and AGADIRIs. The other energy contributions to α-helix stability are not affected by the original statistical approximation. We also discuss the particularities of the model for α-helix formation utilized in AGADIR and compare it with the classical Zimm-Bragg and Lifson-Roig theories. Moreover, we develop the mathematical relationships between the basic AGADIR energy contributions and helix nucleation and elongation, which permit the quantitative comparison between formalisms. Remarkably, the comparison between AGADIRms and the Lifson-Roig formalism shows that, despite the differences on treating helix/coil cooperativity, both theories give virtually identical results when an equivalent set of parameters is used. This indicates that the helix/coil transition is a solid theory independent of the particularities of the model for α-helix formation. © 1997 John Wiley & Sons, Inc. Biopoly 41: 495–509, 1997.     

Zoosporangial discharge in a Protoachlya hypogyna (Saprolegniaceae) isolate from southeastern North Carolina

Inadequate attention to zoosporangial discharge has led to confusion in watermold taxonomic literature. This problem is discussed in light of a specimen of Protoachlya hypogyna that manifests discharge characteristic of three watermold genera, and recommendations are made to reduce future inaccuracies.     

A method on strain measurement of HAP in cortical bone from diffusive profile of X-ray diffraction

Bone tissue is a composite material composed of hydroxyapatite (HAp) and collagen matrix. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the lattice strain of HAp crystals. However, mineral particles of HAp in bone have much lower crystallinity than usual crystalline materials, which show a diffusive intensity profile of X-ray diffraction. It is not easy to determine quantitatively an infinitesimal strain of HAp from the peak position of diffusive profile. In order to improve the accuracy of strain measurement of HAp in bone tissue and to obtain reproducible results, this paper proposes an X-ray diffraction method applied to a diffusive profile for low crystallinity. This method is to estimate the lattice strain of HAp using not a peak position but a whole diffraction profile. In this experiment, a strip specimen of 28 x 8 x 2 mm was made from bone axial, outside circumferential and cross-sectional circumferential region in the cortical bone of bovine femur. The X-ray diffraction measurements were carried out before and after applying an external load. As a result, the precision of strain measurement was much improved by this method. Although a constant value of macroscopic strain was applied in the specimen, the lattice strain had a lower value than the macroscopic strain and had a different value in each specimen.     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

A holistic approach for protein secondary structure estimation from infrared spectra in H(2)O solutions

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique combines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent background subtraction. Previous attempts to generate structural reference spectra from a basis set of reference protein spectra have had limited success because of inaccuracies arising from sequential background subtractions and spectral normalization, arbitrary spectral band truncation, and attempted resolution of spectroscopically degenerate structure classes. We eliminated these inaccuracies by defining a single mathematical function for protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with the sequential nature of previous methods, especially with regard to removing the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unordered helix, sheet, turns, and unordered structures from a basis set of spectra of well-characterized proteins. Structural reference spectra were generated in the amide I and amide III bands, both of which have been shown to be sensitive to protein secondary structure content. We accurately account for overlaps between amide and nonamide regions and allow different structure types to have different extinction coefficients. The agreement between our structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good.     

Heavy water (D2O)-induced shape changes, movements and F-actin redistribution in human neutrophil granulocytes

Heavy water (D2O) induces characteristic shape changes and a distinct type of movement in human neutrophil granulocytes. In contrast to front-tail polarity as evoked by chemotactic peptides and microtubule-disassembling agents, D2O-based media produce non-polar neutrophils with many small or long surface projections. This phenotype is similar to that elicited by both phorbol myristate acetate and diacylglycerols, but the surface projections are smaller and more densely placed and are often associated with a single large projection. D2O-induced non-polar cells with surface projections perform continuous shape changes without front-tail polarity and without the unidirectional movement and cytoplasmic streaming seen in cells with front-tail polarity. Some of the cells show circus movements of a large projection indicating circular polarity. In neutrophils suspended in D2O, F-actin is shifted to the cell periphery, mainly into the surface projections of activated cells. The D2O-induced effects are reversed in H2O-based medium. D2O is dominant over the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), colchicine and taxol in that the combined action of D2O with any of these agents results in the D2O-induced phenotype. In contrast, cytochalasin B alone and in combination with fMLP induces a considerable decrease of non-polar cells and an increase of spherical cells similar to non-stimulated cells in H2O-based medium. Earlier studies indicated that D2O acts on microtubules. Our results suggest that D2O may act on the microfilament system. Neutrophils suspended in D2O-based medium may represent a useful model to study the relationship between shapes, movements, and particular functions of these cells.     

THE NEAR-SPINELESS TRACHELOMONAS GRANDIS (EUGLENOPHYCEAE) SUPERFICIALLY APPEARS SPINY BY ATTRACTING BACTERIA TO ITS SURFACE1,2

Trachelomonas grandis Singh has a mucilaginous, highly porous mineralized lorica (envelope) generally without ornamentation except occasionally for a few short, tapered, anterior or posterior spines. However, as our first cultures of this species aged, rod-shaped structures appeared on the loricas. That these surface projections were bacteria was determined by scanning and transmission electron microscopy. The bacteria, 2-6 μm long with rounded apices, were oriented perpendicular to the exterior lorica surface and were attached on one end by apically produced tie-down fibers. The bacteria also secreted fibers over their entire surface, forming a network between them that collapsed during specimen preparation for scanning electron microscopy. The density of the surface bacteria increased with time until the lorica took on a “spiny” appearance superficially similar to lorica extensions of algal origin. In mature algal specimens, an estimated 1200-1800 bacteria per lorica occurred as a monolayer, the maximum number related to the surface area of the lorica available for bacterial colonization. The bacteria, always motionless while attached, showed putative evidence of budding. Fission formed short chains of up to three cells on the lorica. Our cultures maintained this association for 8 years (1972-1979). However, cultures ordered for further study in the past year have failed to develop loricas with more than just a few bacterial cells, and most have none.     

Analysis of a novel offset cone-beam computed mammotomography system geometry for accomodating various breast sizes

We evaluate a newly developed dedicated cone-beam transmission computed mammotomography (CmT) system configuration using an optimized quasi-monochromatic cone beam technique for attenuation correction of SPECT in a planned dual-modality emission and transmission system for pendant, uncompressed breasts. In this study, we perform initial CmT acquisitions using various sized breast phantoms to evaluate an offset cone-beam geometry. This offset geometry provides conjugate projections through a full 360 degree gantry rotation, and thus yields a greatly increased effective field of view, allowing a much wider range of breast sizes to be imaged without truncation in reconstructed images. Using a tungsten X-ray tube and digital flat-panel X-ray detector in a compact geometry, we obtained initial CmT scans without shift and with the offset geometry, using geometrical frequency/resolution phantoms and two different sizes of breast phantoms. Acquired data were reconstructed using an ordered subsets transmission iterative algorithm. Projection images indicate that the larger, 20 cm wide, breast requires use of a half-cone-beam offset scan to eliminate truncation artifacts. Reconstructed image results illustrate elimination of truncation artifacts, and that the novel quasi-monochromatic beam yields reduced beam hardening. The offset geometry CmT system can indeed potentially be used for structural imaging and accurate attenuation correction for the functional dedicated breast SPECT system.     

Relationship between bone tissue strain and lattice strain of HAp crystals in bovine cortical bone under tensile loading

Cortical bone is a composite material composed of hydroxyapatite (HAp) and collagen. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the strain of HAp crystals. However, HAp crystals in bone tissue have been known to have the low degree of crystallization. Authors have proposed an X-ray diffraction method to measure the lattice strain of HAp crystals from the diffusive intensity profile due to low crystallinity. The precision of strain measurement was greatly improved by this method. In order to confirm the possibility of estimating the bone tissue strain with measurements of the strain of HAp crystals, this work investigates the relationship between bone tissue strain on a macroscopic scale and the lattice strain of HAp crystals on a microscopic scale. The X-ray diffraction experiments were performed under tensile loading. Strip bone specimens of 40x6x0.8mm in size were cut from the cortical region of a shaft of bovine femur. A stepwise tensile load was applied in the longitudinal direction of the specimen. By detecting the diffracted X-ray beam transmitted through the specimen, the lattice strain was directly measured in the loading direction. As a result, the lattice strain of HAp crystals showed lower value than the bone tissue strain measured by a strain gage. The bone tissue strain was described with the mean lattice strain of the HAp crystals and the elastic modulus.     

Method for measuring cardiac dimensions and intramural deformations

Information about the geometry and intramural deformations of the left vertricle of the heart throughout the cardiac cycle is necessary for the analysis of contractile function of the intact heart. In this study a method is developed to measure dimensions and intramural deformations of the left ventricle of a dog heart during the cardiac cycle. Radiopaque markers (platinum spheres of 1mm diameter) were implanted in the wall of the left ventricle. The spatial positions of these markers were determined from pairs of perpendicular X-ray projections, obtained with the aid of rotational X-ray equipment. Preliminary results of venticular deformations are in agreement with measurements by other techniques.     

Central projections of peripheral mechanosensory cells with increased excitability in Drosophila mosaics

The formation and maintenance of the central projections of identified bristle mechanosensory neurons with altered excitability were examined in Drosophila mosaics. Two mutants, eag (ether à go-go) and Sh (Shaker), are known to increase excitability of both nerve and muscle cells and enhance synaptic transmission by affecting different types of K+ currents. The eag Sh double mutant produces a synergistic effect, resulting in a greatly increased level of spontaneous neuronal activity and extreme behavioral phenotypes. By constructing mosaic flies containing small patches of doubly mutant cuticle, it was possible to alter the excitability of only one or two identified sensory cells without affecting the surrounding tissue. In these mosaic flies, the doubly mutant sensory cells were more responsive to tactile stimulation. A CoCl2 backfilling technique was utilized in staining the sensory cell projections. Both qualitative and quantitative comparisons were made between projections of cells having normal and increased levels of excitability. The length, branching characteristics, and number of terminal varicosities were analyzed for each sensory cell projection. Results indicate that, at the light microscopy level, these characteristics were not obviously altered by an increased level of excitability.     

Considerations on the thalamostriatal system with some functional implications

The thalamostriatal projections are largely neglected in current reviews dealing with basal ganglia function. In the past few years, however, several studies have re-evaluated these projections and have postulated their implication in more complex tasks within the basal ganglia organization. In this review, we try to focus on the morphological and functional importance of this system in the basal ganglia of the rat, cat and monkey. Special attention is paid to the thalamus as an important place for interaction between the input and the output systems of the basal ganglia through the thalamostriatal projections. Thus, we stress on the overlapping thalamic territories between the thalamic projection of the output nuclei of the basal ganglia and the thalamostriatal neurons. Our experimental data support the existence of several thalamic feedback circuits within the basal ganglia functional design. Finally, some considerations are provided upon the functional significance of these thalamic feedback circuits in the overall organization of the basal ganglia in health and disease.     

Exposures in interventional radiology using Monte Carlo simulation coupled with virtual anthropomorphic phantoms

In this work we investigated the way in which conversion coefficients from air kerma-area product for effective doses (CC_E) and entrance skin doses (CC_ESD) in interventional radiology (IR) are affected by variations in the filtration, projection angle of the X-ray beam, lead curtain attached to the surgical table, and suspended shield lead glass in regular conditions of medical practice. Computer simulations were used to model an exposure scenario similar to a real IR room. The patient and the physician were represented by MASH virtual anthropomorphic phantoms, inserted in the MCNPX 2.7.0 radiation transport code. In all cases, the addition of copper filtration also increased the CC_E and CC_ESD values. The highest CC_E values were obtained for lateral, cranial and caudal projections. In these projections, the X-ray tube was located above the table, and more scattered radiation reached the middle and upper portions of the physician trunk, where most of the radiosensitive organs are located. Another important result of this study was to show that the physician's protection is 358% higher when the lead curtain and suspended shield lead glasses are used. The values of CC_E and CC_ESD, presented in this study, are an important resource for calculation of effective doses and entrance skin doses in clinical practice.     

Analysis of the hierarchical structure of biological tissues by scanning X-ray scattering using a micro-beam.

The outstanding mechanical properties of biological tissues such as wood or bone are mainly due to their hierarchical structure and to their optimization at all levels of hierarchy. It is therefore essential to characterize the structure at all levels to understand the complex behavior of such tissues. Structures down to the micrometer level are accessible to light or scanning electron microscopic observation. In the case of bone this includes, for example, morphometry of the trabecular architecture or the bone mineral density distribution in cortical and trabecular bone. To characterize the sub-micrometer structure of, e.g., the collagen-mineral composite in the case of bone or the cellulose microfibrils in the case of wood, other methods, such as transmission electron microscopy or X-ray scattering are necessary. The recent availability of extremely brilliant synchrotron X-ray sources has led to the development of the new techniques of scanning small-angle X-ray scattering and scanning X-ray microdiffraction, which are capable of providing structural information on the micrometer and the nanometer level, simultaneously. As a basic principle of the method the specimen is scanned across an X-ray beam which has a diameter of few micrometers. Measuring the X-ray absorption at each position provides an image of the specimen (microradiography) with resolution similar to light microscopy, in the micrometer range. Moreover, the X-ray scattering pattern is analyzed at each specimen position to provide parameters characterizing the structure in the nanometer range. The present paper reviews the principles of the techniques and demonstrates their application to biological materials, such as wood or bone.     

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

Keeping in check painful synapses in central amygdala

Glutamatergic projections from the parabrachial nucleus to the central amygdala are implicated in pain transmission. In this issue of Neuron, Delaney et al. identify a new form of adrenergic modulation at these synapses, demonstrating that noradrenaline-induced suppression of glutamate release is mediated by a decrease in the number of sites of synaptic transmission without changes in probability of release.     

Lipovitellin-phosvitin crystals with orthorhombic features: thin-section electron microscopy, gel electrophoresis, and microanalysis in teleost and amphibian yolk platelets and a comparison with other vertebrates

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.