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PubMed2001

Virtual endoscopy of the tympanic cavity based on high-resolution multislice computed tomographic data.

OBJECTIVE: This study was designed to assess the value of high-resolution multislice computed tomography (MSCT) data of the petrous bone for the virtual endoscopic visualization of the tympanic cavity. BACKGROUND: The recently introduced MSCT technology has improved spatial resolution in the z axis as well as scan speed in computed tomography. Three-dimensional rendering of high-resolution MSCT data of the petrous bone may be expected to provide endoluminal views of superior image quality, thus competing with transtympanic endoscopy (otoendoscopy). SETTING: This study was conducted at a university teaching hospital. MATERIALS AND METHODS: Cadaveric phantom studies in a MSCT scanner were performed to define a data acquisition protocol, combining adequate detail resolution with low tube current. Subsequently, the cadaveric phantom underwent otoendoscopy. The postprocessing parameters of the three-dimensional rendering protocol were chosen to produce views closely resembling the corresponding otoendoscopic images. High-resolution data from 18 patients with pathologic conditions of the middle ear, as suggested by clinical findings and assessment of cross-sectional data, were postprocessed using the volume rendering technique to generate standardized virtual endoscopic views. A total of 36 virtual endoscopic scans of the tympanic cavity were generated. RESULTS: With regard to intermediate and high-density structures, virtual endoscopic images, based on MSCT data, yielded endoluminal views closely resembling corresponding otoendoscopic views. Virtual endoscopy seems useful for imaging ossicular pathologic conditions such as dysplasia and chain disruption as well as for assessing patient status before and after otosurgery. CONCLUSION: MSCT data sets allow for generating virtual endoscopic views closely resembling otoendoscopic images. The technique is especially useful when ossicular pathologic changes are present as well as for preoperative and postoperative imaging of otologic procedures.

Endoscopy↗

PubMed2003

Diffusivity- and T2 imaging at 3 Tesla for the detection of degenerative changes in human-excised tissue with high resolution: atherosclerotic arteries.

RATIONALE AND OBJECTIVES: We investigated whether it is possible to investigate degenerative changes in human tissue on a sub-100-microm resolution scale not only on special high-field small-bore MR-microscopy systems but also on a 3T whole-body MR-scanner. METHODS: Spin-spin relaxation, proton density, and diffusion microimaging were investigated in studying human atherosclerotic arteries. Strong diffusion weighting and high spatial resolution was achieved by means of a strong dedicated gradient system and a small birdcage radiofrequency resonator. RESULTS: Quantitative parameter maps were obtained at voxel sizes down to 73 x 73 x 600 microm3. The morphologic structure and pathology connected to lipid deposits, plaques, small thrombi, and bifurcations were well visualized. CONCLUSION: High-resolution parameter-weighted and parameter-imaging at sub-100-microm pixel resolution can be achieved for excised tissue on a 3.0 T whole body MR system. Perspectives for the characterization of atherosclerotic plaques imply not only cost advantages but also equivalence of contrast, especially as to T(2), for in vivo and high-resolution ex vivo investigations on the same MR scanner.

Aged↗

PubMed2006

Influence of heart rate and temporal resolution on left-ventricular volumes in cardiac multislice spiral computed tomography: a phantom study.

PURPOSE: We sought to investigate the influence of heart rate and temporal resolution on the assessment of left-ventricular (LV) function with multislice spiral computed tomography (CT). MATERIAL AND METHODS: A dynamic cardiac phantom was repeatedly scanned with a 64-slice CT scanner using a standardized scan protocol (64 x 0.6 mm, 120kV, 770mAs(eff), 330 milliseconds rotation time) at different simulated heart rates, ranging from 40 to 140 beats per minute. Images were reconstructed with an algorithm utilizing data from 1 to 4 cardiac cycles (RR intervals). Ejection fraction (EF), end-systolic, end-diastolic, and stroke volume as well as cardiac output were calculated. Results of the measurements were compared with the real volumes of the phantom. Interscan and intraobserver variability were calculated. RESULTS: Using a monosegmental reconstruction algorithm, the temporal resolution was fixed to 165 milliseconds. With bi-, tri-, and quad-segmental image reconstruction, mean temporal resolution was 128.3 +/- 33.2 milliseconds, 103.3 +/- 49.2 milliseconds, and 87.8 +/- 81.5 milliseconds, respectively. Multisegmental image reconstruction resulted in a lower deviation when comparing measured and real volumes. Using mono-, bi-, tri-, and quad-segmental image reconstruction, the percent deviation between measured and real values for EF was 8.2%, 4.5%, 3.3%, and 3.4%, respectively. Applying multisegmental image reconstruction with improved temporal resolution the deviation decreased with increasing heart rate when compared with mono-segmental image reconstruction. Interscan and intraobserver variability for EF were 1.1% and 1.9%, respectively. CONCLUSION: Enhanced temporal resolution improves the quantification of LV volumes in cardiac multislice spiral CT, enabling reliable assessment of LV volumes even at increased heart rates.

Algorithms↗

PubMed2002

Time-resolved diffraction and interference: Young's interference with photons of different energy as revealed by time resolution.

We present time-resolved diffraction and two-slit interference experiments using a streak camera as a detector for femtosecond pulses of photons. These experiments show how the diffraction pattern is built by adding frames of a few photons to each frame. It is estimated that after 300 photons the diffraction pattern emerges. With time resolution we can check the speed of light and put an upper limit of 2 ps at our resolution to the time for wave function collapse in the quantum measurement process. We then produce interference experiments with photons of different energies impinging on the slits, i.e. we know which photon impinges on each slit. We show that for poor time resolution, no interference is observed, but for high time resolution, we have interference that is revealed as beats of 100 GHz frequency. The condition for interference is that the two pulses should overlap spatially at the detector, even if the pulses have different energies but are generated from the same pulse of the laser. The interference seems to be in agreement with classical theory at first sight. However, closer study and analysis of the data show deviations in the visibility of the interference fringes and of their phase. These experiments are discussed in connection with quantum mechanics and it may be concluded that the time resolution provides new data for understanding the longstanding and continuing arguments on wave-particle duality initiated by Newton, Young, Fresnel, Planck and others. A thought experiment is presented in the appendix to try to distinguish the photons at the detector by making it sensitive to colour.

Energy Transfer↗

PubMed1999

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of holliday junction resolution.

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.

Bacterial Proteins↗

PubMed2002

High resolution 3D x-ray diffraction microscopy.

We have imaged a 2D buried Ni nanostructure at 8 nm resolution using coherent x-ray diffraction and the oversampling phasing method. By employing a 3D imaging reconstruction algorithm, for the first time we have experimentally determined the 3D structure of a noncrystalline nanostructured material at 50 nm resolution. The 2D and 3D imaging resolution is currently limited by the exposure time and the computing power, while the ultimate resolution is limited by the x-ray wavelengths. We believe these results pave the way for the development of atomic resolution 3D x-ray diffraction microscopy.

Microscopy↗

PubMed2001

On the use of low-resolution data for translation search in molecular replacement.

Low-resolution reflections (approximately 15 A and lower) are very useful for the translation search in molecular replacement because they are less sensitive to model errors compared with the traditionally used reflections of resolution 4-10 A. At low resolution, however, the contribution from the bulk solvent is quite significant and corresponding structure factors calculated from a macromolecular model cannot be compared with experimental values if this contribution is neglected. The proposed method provides a way of fast translation searches where low-resolution reflections are taken into account. Test calculations using several experimental data sets show a dramatic improvement in the signal after the bulk-solvent correction and low-resolution reflections were included in the calculation; this improvement allowed unambiguous identification of the solution.

Algorithms↗

PubMed2000

Globbic approximation in low-resolution direct-methods phasing.

Probabilistic direct-methods phasing theory, originally based on a uniform atomic distribution hypothesis, is shown to be adaptable to a non-uniform bulk-solvent-compensated globbic approximation for protein crystals at low resolution. The effective number n(g) of non-H protein atoms per polyatomic glob increases with decreasing resolution; low-resolution phases depend on the positions of only N(g) = N(a)/n(g) globs rather than N(a) atoms. Test calculations were performed with measured structure-factor data and the refined structural parameters from a protein crystal with approximately 10 000 non-H protein atoms per molecule and approximately 60% solvent volume. Low-resolution data sets with d(min) ranging from 15 to 5 A gave n(g) = ad(min) + b, with a = 1.0 A(-1) and b = -1.9 for the test case. Results of tangent-formula phase-estimation trials emphasize that completeness of the low-resolution data is critically important for probabilistic phasing.

Crystallization↗

PubMed2000

Low-resolution phase information in multiple-wavelength anomalous solvent contrast variation experiments.

The basic theory and principles of the multiple-wavelength anomalous solvent-contrast (MASC) method are introduced as a contrast-variation technique for generating low-resolution crystallographic phase information on the envelope of a macromolecule. Experimental techniques and practical considerations concerning the choice of anomalous scatterer, sample preparation and data acquisition are discussed. Test cases of crystals of three proteins of differing molecular weights from 14 kDa through to 173 kDa are illustrated. Methods for extracting the moduli of the anomalous structure factors from the MASC data are briefly discussed and the experimental results are compared with the known macromolecular envelopes. In all cases, the lowest resolution shells exhibit very large anomalous signals which diminish at higher resolution, as expected by theory. However, in each case the anomalous signal persists at high resolution, which is strong evidence for ordered sites of the anomalous scatterers. For the smaller two of these proteins the heavy-atom parameters could be refined for some of these sites. Finally, a novel method for phasing the envelope structure-factor moduli is presented. This method takes into account the relatively low number of observations at low resolution and describes the macromolecular envelope with a small number of parameters by presuming that the envelope is a compact domain of known volume. The parameterized envelope is expressed as a linear combination of independent functions such as spherical harmonics. Phasing starts from solutions of a sphere in the unit cell after positional refinement from random trials and the parameters describing the envelope are then refined against the data of structure-factor moduli. The preliminary results using simulated data show that the method can be used to reconstruct the correct macromolecular envelope and is able to discriminate against some false solutions.

Aldose-Ketose Isomerases↗

PubMed2004

A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolution.

Xylanases (1,4-beta-D-xylan xylanhydrolases; EC 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C-centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1 A, beta = 119.7 degrees. These crystals diffract X-rays to better than 1.5 A resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45 A resolution (94.1% completeness, R(merge) = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95 K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8 A resolution diffraction data at 100 K (90.9% completeness; R(merge) = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability.

Bacterial Proteins↗

PubMed2006

Hydrophobin HFBII in detail: ultrahigh-resolution structure at 0.75 A.

Hydrophobins are small proteins secreted by filamentous fungi that have a unique ability to spontaneously form amphiphilic layers. Hydrophobins have only recently been structurally characterized through the first crystal structure determination of a protein of this class, Trichoderma reesei hydrophobin HFBII [Hakanpää, Paananen et al. (2004), J. Biol. Chem. 279, 534-539]. The resolution of the HFBII structure has now been extended to an ultrahigh resolution of 0.75 A. The structure was refined conventionally and multipole refinement has been initiated. The ultrahigh-resolution structure is analyzed here in detail and comparison is made to the previous atomic resolution structure of the same protein as well as to other ultrahigh-resolution structures found in the Protein Data Bank.

Anisotropy↗

PubMed1993

High-resolution refinement of the hexagonal A-DNA octamer d(GTGTACAC) at 1.4 A.

The hexagonal crystal form of the octamer d(GTGTACAC), grown in the presence of spermine, has unit-cell dimensions a = b = 32.18 and c = 78.51 A, space group P6(1)22, with one DNA strand in the asymmetric unit. The structure has been refined starting with the earlier lower resolution model and using high-resolution 1.4 A data collected on a Siemens-Xentronics area detector at 258 K. There were 4365 unique reflections greater than 2sigma(F) in the resolution range 5-1.4 A. The model was refitted into 3F(o) - 2F(c). Sim-weighted omit maps and difference maps were used to locate water molecules. The final model with 161 DNA atoms and 37 water molecules gave an R factor of 19.8%. Crystals of the same octamer were also grown in the presence of spermidine instead of spermine, and refinement using nominal 1.45 A resolution data, 3292 unique reflections, final R = 19.1%, gave virtually identical DNA parameters. No bound spermine or spermidine was detected in either of these structure analyses. The electron density was clear for the DNA and showed holes in the center of the six-membered rings of bases, and also in the center of some of the sugar rings. The high-resolution structure has provided more precise DNA parameters and confirmed the features observed in the earlier 2 A study including the packing-induced distortion in the A7 (A15) sugar pucker from C(3')-endo and C(2')-endo. This change causes the end base pairs to bend away from the helix axis while the rest of the duplex is nearly linear. The hydration patterns in the deep and shallow grooves have been characterized. Chains of water molecules were found, but no rings. The familiar intermolecular contact region between the end base pair and the minor groove of a symmetry-related duplex, involving four residues on one strand and two on the other, has been analyzed. One of these interactions is a hydrogen bond.

Journal Article↗

PubMed1996

Refined structure of the chitinase from barley seeds at 2.0 a resolution.

Chitinase from barley seeds is a monomeric enzyme with 243 amino-acid residues and it plays a role as a defense protein. Its structure, previously determined at 2.8 A resolution by multiple isomorphous replacement method, is mainly alpha-helical [Hart, Monzingo, Ready, Ernst & Robertus, (1993). J. Mol. Biol. 229, 189-193]. The crystallization and preliminary X-ray data of the same enzyme in a different crystal form has been reported independently [Song, Hwang, Kim & Suh, (1993). Proteins, 17, 107-109}, the asymmetric unit of which contains two chitinase molecules. As a step toward understanding the general principles of catalysis, reported here is the structure of chitinase from barley seeds in this crystal form, as determined by molecular replacement and subsequently refined at 2.0 A resolution, with incorporation of partial data to 1.9 A (R factor of 18.9% for 31 038 unique reflections with F(o)> 2sigma(F) in the range 8.0-1.9 A). The r.m.s. deviations from ideal stereochemistry are 0.013 A for bond lengths and 1.32 degrees for bond angles. A superposition of the two independent molecules in the asymmetric unit gives an r.m.s. difference of 0.55 A for all protein atoms (0.43 and 0.74 A for main-chain and side-chain atoms, respectively). When the refined model of each chitinase molecule in the asymmetric unit is superposed with the starting model, the r.m.s. difference for all shared protein atoms is 0.99 A for molecule 1 and 0.85 A for molecule 2, respectively. Through a sequence comparison with homologous plant chitinases as well as a structural comparison with the active sites of other glycosidases, key catalytic residues have been identified and the active site has been located in the three-dimensional structure of the barley chitinase. The present structure, refined at an effective resolution of 2.0 A with incorporation of partial data to 1.9 A, represents a significant improvement in resolution compared to the previously reported model. The improved resolution has enabled the location of solvent atoms, including water molecules near the catalytic residues, in addition to the positioning of protein atoms with greater accuracy.

Journal Article↗

PubMed1999

The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus.

Protein HU is a ubiquitous prokaryotic protein which controls the architecture of genomic DNA. It binds DNA non-specifically and promotes the bending and supercoiling of the double helical structure. HU is involved in many DNA-associated cellular processes, including replication, transcription and the packaging of DNA into chromosome-like structures. Originally determined at medium resolution, the crystal structure of HU has now been refined at 2.0 A resolution. The high-resolution structure shows that the dimeric molecule is essentially a compact platform for two flexible and basic arms which wrap around the DNA molecule. To maximize the protein's stability, non-secondary structural regions are reduced to a minimum, there is an extensive aromatic hydrophobic core and several salt bridges and hydrogen-bonded water molecules knit together crucial regions. Based on the original medium-resolution structure of HU, several proposals were made concerning the structural basis of HU's ability to bind, bend and supercoil DNA. Each of these proposals is fully supported by the high-resolution structure. Most notably, the surfaces of the molecule which appear to mediate protein-DNA and protein-protein interactions have the ideal shapes and physicochemical properties to perform these functions.

Bacterial Proteins↗

PubMed2003

Applications of ACORN to data at 1.45 A resolution.

One of the main interests in the molecular biosciences is in understanding structure-function relations and X-ray crystallography plays a major role in this. ACORN can be used as a comprehensive and efficient phasing procedure for the determination of protein structures when atomic resolution data are available. An initial model can automatically be built by ARP/wARP followed by REFMAC for refinement. The alpha helices and beta sheets occurring in many protein structures can be taken as starting fragments for structure solution in ACORN. ACORN, along with ARP/wARP followed by REFMAC, can be an ab initio method for solving protein structure for which data are better than 1.2 A (atomic resolution). Attempts are here made in extending its applications to real data at 1.45 A resolution and also to truncated data at 1.6 A resolution. Two previously known structures, congerin II and alkaline cellulase N257, were resolved using the above approach. Automatic structure solution, phasing and refinement for real data at still lower resolutions for proteins of various complexities are being carried out. Data mining of the secondary structural features using PDB is being carried out for this new approach for 'seed-phasing' to ACORN.

Algorithms↗

PubMed2005

Spherically bent analyzers for resonant inelastic X-ray scattering with intrinsic resolution below 200 meV.

Resonant inelastic X-ray scattering with very high energy resolution is a promising technique for investigating the electronic structure of strongly correlated materials. The demands for this technique are analyzers which deliver an energy resolution of the order of 200 meV full width at half-maximum or below, at energies corresponding to the K-edges of transition metals (Cu, Ni, Co etc.). To date, high resolution under these conditions has been achieved only with diced Ge analyzers working at the Cu K-edge. Here, by perfecting each aspect of the fabrication, it is shown that spherically bent Si analyzers can provide the required energy resolution. Such analyzers have been successfully produced and have greatly improved the energy resolution in standard spherically bent analyzers.

Equipment Design↗

PubMed1998

An evaluation of exact and approximate 3-D reconstruction algorithms for a high-resolution, small-animal PET scanner.

MicroPET is a low-cost, high-resolution positron emission tomography (PET) scanner designed for imaging small animals. MicroPET operates exclusively without septa, acquiring fully three-dimensional (3-D) data sets. The performance of the projection-reprojection (3DRP), variable axial rebinning (VARB), single slice rebinning (SSRB), and Fourier rebinning (FORE) methods for reconstruction of microPET data were evaluated. The algorithms were compared with respect to resolution, noise variance, and reconstruction time. Results suggested that the 3DRP algorithm gives the best combination of resolution and noise performance in 9 min of reconstruction time on a Sun UltraSparc I workstation. The FORE algorithm provided the most acceptable accelerated method of reconstruction, giving similar resolution performance with a 10%-20% degradation in noise variance in under 2 min. Significant degradation in the axial resolution was measured with the VARB and SSRB methods, offsetting the decrease in reconstruction time achieved with those methods. In-plane angular mashing of the 3-D data before reconstruction led to a 50% reduction in reconstruction time but also introduced unacceptable tangential blurring artifacts. This thorough evaluation of analytical 3-D reconstruction techniques allowed for optimal selection of a reconstruction method for the diverse range of microPET applications.

Algorithms↗

PubMed2005

Estimation of the cortical connectivity by high-resolution EEG and structural equation modeling: simulations and application to finger tapping data.

Today, the concept of brain connectivity plays a central role in the neuroscience. While functional connectivity is defined as the temporal coherence between the activities of different brain areas, the effective connectivity is defined as the simplest brain circuit that would produce the same temporal relationship as observed experimentally between cortical sites. The most used method to estimate effective connectivity in neuroscience is the structural equation modeling (SEM), typically used on data related to the brain hemodynamic behavior. However, the use of hemodynamic measures limits the temporal resolution on which the brain process can be followed. The present research proposes the use of the SEM approach on the cortical waveforms estimated from the high-resolution EEG data, which exhibits a good spatial resolution and a higher temporal resolution than hemodynamic measures. We performed a simulation study, in which different main factors were systematically manipulated in the generation of test signals, and the errors in the estimated connectivity were evaluated by the analysis of variance (ANOVA). Such factors were the signal-to-noise ratio and the duration of the simulated cortical activity. Since SEM technique is based on the use of a model formulated on the basis of anatomical and physiological constraints, different experimental conditions were analyzed, in order to evaluate the effect of errors made in the a priori model formulation on its performances. The feasibility of the proposed approach has been shown in a human study using high-resolution EEG recordings related to finger tapping movements.

Algorithms↗