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PubMed2003

Improvement of optical resolution in far-field imaging by optical multiplication.

Based on the idea of the complex filters suggested by Toraldo di Francia in 1952, super-resolved imaging has been achieved by using interferometric image multiplication. The question of whether super-resolution implies a limited field of view is discussed, and an analytical criterion to decide this question is obtained. Three different imaging configurations are investigated, all of which gave a spatial resolution exceeding that corresponding to the full open aperture of the optical system. A resolution limit 55% of the Rayleigh limit was achieved for incoherently illuminated objects. One price that must be paid for the super-resolution is inefficient use of the light source. A second price is a limitation to the usable field of view.

Fourier Analysis↗

PubMed2000

High resolution transmission electron microscopy to study very thin crystalline layers buried at an amorphous-crystalline interface

Structure characterisation of interfaces is a field of widespread application of high resolution transmission electron microscopy for its very high spatial resolution. Specimen thickness and electron optical condition have a deep influence on the high resolution electron transmission microscopy image contrast. Hence, in many cases, the real structure of the sample can be understood from experimental images only by comparison with the relevant simulation. Moreover, the understanding of the contrast variation of a few A at an interface is a task in which even the use of simulation could not produce an unequivocal solution of the experimental result. In this paper high resolution transmission electron microscopy image simulations show that two monolayers of crystalline material buried at an amorphous-crystalline interface can be successfully revealed and interpreted. The simulated images reproduce the experimental results as obtained from the Al/Si-As/n-GaAs (001) heterostructure.

Journal Article↗

PubMed2000

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome.

BACKGROUND: This study addresses the general problem of dividing a density map of a nucleic-acid-protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches approximately 3 A it is generally possible to interpret its shape (i. e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. RESULTS: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 A resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. CONCLUSIONS: The new method, when applied to an 11.5 A cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.

Bacterial Proteins↗

PubMed1997

High-resolution polypeptide structure in a lamellar phase lipid environment from solid state NMR derived orientational constraints.

BACKGROUND: Solid-state nuclear magnetic resonance (NMR) spectroscopy provides novel structural constraints from uniformly aligned samples. These orientational constraints orient specific atomic sites with respect to the magnetic field direction and the unique molecular axis of alignment. Solid-state NMR is uniquely and ideally suited for providing such structural constraints on polypeptides and proteins in a lamellar phase lipid environment. Membrane protein structure represents a great challenge for structural biologists; a new approach for characterizing high resolution three-dimensional structure in such an environment is needed. RESULTS: The optimal use of orientational constraints for defining three-dimensional structures is demonstrated with the elucidation of the gramicidin A channel structure at high resolution. Initial structures are refined against both the experimental constraints and the CHARMM energy using a novel simulated-annealing protocol to define torsion angle solutions with an error bar of approximately +/- 5 degrees. CONCLUSIONS: This analysis results in the determination of a high-resolution, time averaged structure of gramicidin A obtained in a lipid bilayer environment above the gel-to-liquid crystalline phase transition temperature. It is demonstrated that solid-state NMR can be used to establish polypeptide, and potentially protein, structures in such an environment. Furthermore, this high-resolution structure is demonstrated to provide new insights into polypeptide function. For the gramicidin A channel the roles of the indole groups that facilitate ion transport and details of the cation solvation environment provided by the amide oxygens are characterized.

Amino Acid Sequence↗

PubMed2002

Mass correlated acceleration in a reflectron MALDI TOF mass spectrometer: an approach for enhanced resolution over a broad mass range.

Compared to continuous extraction, pulsed extraction (PE) of ions formed by matrix-assisted laser desorption/ionization (MALDI) in time-of-flight (TOF) mass spectrometers significantly improves mass resolution. Parameters such as extraction voltage, delay time, and correction pulse must be varied, however, to achieve optimum mass resolution over a broad mass range because the PE method is mass dependent. We previously reported a novel method, mass correlated acceleration (MCA), which we have now combined with a reflectron MALDI TOF mass spectrometer to further enhance mass resolution over a broader mass range. Unlike the PE method, MCA is not mass dependent and high resolution mass spectra can be achieved with a single tuning of instrument parameters. The ions may be brought into focus simultaneously, i.e., the multi-channel recording advantage can be more fully realized. The MCA dual-stage ion source design includes an extraction pulse region and an acceleration region that contains a time-dependent waveform correlated with mass. We demonstrate the validity of this novel technique with applications in peptide mixture analysis and protein digests of lysozyme and bovine serum albumin.

Algorithms↗

PubMed2002

IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database.

Recent advances in electron cryomicroscopy instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3D) structure determination of macromolecular complexes. However, it has become impractical and inefficient to rely on conventional text file data management and command-line programs to organize and process the increasing numbers of image data required in high-resolution studies. Here, we present a distributed relational database for managing complex datasets and its integration into our high-resolution software package IMIRS (Image Management and Icosahedral Reconstruction System). IMIRS consists of a complete set of modular programs for icosahedral reconstruction organized under a graphical user interface and provides options for user-friendly, step-by-step data processing as well as automatic reconstruction. We show that the integration of data management with processing in IMIRS automates the tedious tasks of data management, enables data coherence, and facilitates information sharing in a distributed computer and user environment without significantly increasing the time of program execution. We demonstrate the applicability of IMIRS in icosahedral reconstruction toward high resolution by using it to obtain an 8-A 3D structure of an intermediate-sized dsRNA virus.

Animals↗

PubMed2000

Diagnostic imaging with newly developed ortho cubic super-high resolution computed tomography (Ortho-CT).

OBJECTIVE: Ortho cubic super-high resolution computed tomography (Ortho-CT), which we have developed, is characterized by the small size of the unit and the ability to produce 3-dimensional images of high resolution with low-radiation doses. The purpose of this study was to investigate the clinical usefulness of Ortho-CT for the diagnosis of diseases in the oral and maxillofacial region. STUDY DESIGN: Ortho-CT has been used for the imaging diagnosis of a radicular cyst of the upper first molar, mesiodense, tooth fractures in the upper anterior region, a fracture of the condylar process, or presurgical evaluation for a dental implant. The usefulness of 3-dimensional images obtained by Ortho-CT was investigated in this study. RESULTS: Ortho-CT produced images of high resolution, enabling identification of the extent of the lesions and the fracture line, the condition of the impacted tooth, and the relationship with the maxillary sinus, mandibular canal, and adjacent teeth. CONCLUSION: Because Ortho-CT can take high-resolution 3-dimensional images at any tomographic layer with only 1 exposure, it is useful for the diagnosis of diseases in the oral and maxillofacial region.

Adult↗

PubMedDate not supplied

High resolution 2D 1H-13C correlation of cholesterol in model membrane.

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.

Cholesterol↗

PubMed2002

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

One-second time resolution brain microdialysis in fully awake rats. Protocol for the collection, separation and sorting of nanoliter dialysate volumes.

Capillary zone electrophoresis is capable of analyzing nanoliter volumes, reducing the challenge posed by brain microdialysis time resolution improvement to the management of nanoliter dialysate volumes. This fact has not been overlooked and 12- and 6-s time resolution microdialysis have been reported in anesthetized rats. However, behavioral experiments require fully awake and freely moving animals. To achieve high temporal resolution brain microdialysis in awake unrestrained rats, we have developed an online device that mixes the outflowing dialysate with fluorescein isothiocyanate and buffer within a 26-nl reactor. The mixture was continuously accumulated in a 99-micrometer-bore capillary tube. After the experiment the tube was cut into 4-mm pieces and the content of each piece (30 nl, equivalent to 1 s dialysate) was transferred to a test tube. After allowing 18 h for derivatization, the samples were diluted with water and injected into a capillary electrophoresis laser-induced fluorescence detection instrument. This protocol was tested first in an in vitro assay and proved to be capable of detecting glutamate concentration changes in only 1 s. For the in vivo assays, a probe was inserted into the primary somatosensory cortex of eight rats divided in two groups. One group was stimulated by gently moving its whiskers for 10 s. The other group had no whisker manipulation. Moving the whiskers released glutamate in the experimental group. The first and only change was observed at the 12th s. This method allows 1-s time resolution brain microdialysis in freely moving rats and multiple amino acid analysis every second during sensory perception or motor actions in behavioral experiments.

Animals↗

PubMed1998

Mechanical properties of ewe vertebral cancellous bone compared with histomorphometry and high-resolution computed tomography parameters.

The goal of the present study was to determine if a high-resolution computed tomography (HRCT) system with 150 microns resolution was sufficient to predict mechanical properties in ewe lumbar vertebrae. To answer this question, we used a triangular comparison between: HRCT; biomechanics (compression and shear tests); and histomorphometry, which was the reference method for the measurements of morphometric parameters. Two dissected lumbar vertebrae (L-4 and L-5) from 32 ewes were used. Both compressive and shear properties correlated significantly with amount of bone and structural parameters evaluated by histomorphometry (bone volume/tissue volume, trabecular thickness, trabecular separation), but no significant correlation was found with the trabecular number. With our shear test involving the trabecular architecture itself more significant correlations were found with the node-strut analysis parameters than from the compressive test. Significant correlations were also found between HRCT and histological parameters (bone volume/tissue volume, bone surface/bone volume, trabecular separation, trabecular number, total strut length, number of nodes, and number of termini). Correlations between HRCT structural parameters and mechanical properties on L-4 were of the same magnitude as the correlations between the histomorphometric structural parameters and mechanical results on L-5 but with the remarkable advantage the HRCT is a noninvasive method. In spite of the resolution (150 microns) of our HRCT system, which entailed mainly an enlargement of the thinnest trabeculae or their loss during the segmentation process, we obtained coherent relationships between mechanical and tomographic parameters. The thinnest trabeculae probably had little effect on the mechanical strength. Also, this type of resolution allows us to consider the possibility of perfecting an in vivo HRCT system. However, physical density and bone mineral density correlated much better with strength than either classical histomorphometric or tomographic parameters. The current conclusion is fairly negative with respect to the ability of HRCT to assess mechanical properties nondestructively as compared with dual-energy X-ray absorptiometry. But, the noninvasive nature of the imaging modality and the capacity for three-dimensional imaging at arbitrary orientation make HRCT a promising tool in the quantitative assessment of cancellous architecture.

Animals↗

PubMed2000

Reduced visual resolution acuity and cerebral white matter damage in very-low-birthweight infants.

Neonatal cerebral white matter echolucencies predict visual resolution acuity deficits in very-low-birthweight (VLBW) infants. We examined maternal sociodemographic, lifestyle, intrapartum, infant birth/perinatal, and ocular motor/refractive characteristics to determine whether they accounted for this association in infants who were tested once between postnatal age 25 and 56 weeks (corrected for gestational age at birth). Cranial ultrasound scans were read by consensus to identify echolucency in a population of VLBW infants with no known ocular abnormalities. Visual resolution acuity was measured with the Acuity Card Procedure (ACP) in 14 infants with echolucency and compared with that of 81 VLBW infants born in the same hospitals with normal ultrasound scans. In time-oriented logistic regression models, echolucency remained a consistent predictor of abnormal visual resolution acuity after adjustment for covariates in three developmental periods (pre-, peri-, and postnatal). Odds ratios ranged from 19.3 (95% confidence interval, 4.5 to 82.2; p=0.001) to 10.4 (95% confidence interval, 1.3 to 81.9; p=0.03). Reduced visual resolution acuity in VLBW infants appears to be due to cerebral white matter damage.

Cerebral Cortex↗

PubMed2003

High resolution CryoFESEM of microbial surfaces.

The outer surfaces of three microorganisms, Giardia lamblia, Enterococcus faecalis, and Proteus mirabilis, were investigated by cryo-immobilization followed by sublimation of extracellular ice and cryocoating with either Pt alone or Pt plus carbon. Cryocoated samples were examined at -125 degrees C in either an in-lens field emission SEM or a below-the-lens field emission SEM. Cryocoating with Pt alone was sufficient for low magnification observation, but attempts to do high-resolution imaging resulted in radiolysis and cracking of the specimen surface. Double coating with Pt and carbon, in combination with high resolution backscatter electron detectors, enabled high-resolution imaging of the glycocalyx of bacteria, revealing a sponge-like network over the surface. High resolution examination of bacterial flagella also revealed a periodic substructure. Common artifacts included radiolysis leading to "cracking" of the surface, and insufficient deposition of Pt resulting in the absence of detectable surface topography.

Animals↗

PubMed2000

Improvement of resolution, mass accuracy, and reproducibility in reflected mode DE-MALDI-TOF analysis of DNA using fast evaporation--overlayer sample preparations.

DNA analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is hindered by two processes: alkali metal adduction and fragmentation of the intact ionized molecule. The adverse effects of both processes can be reduced by adding ammonium ion salts or compounds such as fructose to the sample preparations. Matrix additives improve sensitivity and resolution of DNA analysis by MALDI. In addition, spot-to-spot reproducibility, resolution, and mass accuracy for DNA oligonucleotides (< or = 12 mer) can be improved by the use of overlayer sample preparations with matrixes that have low aqueous solubilities, such as alpha-cyano-4-hydroxycinnamic acid, ferulic acid, and 2,4,6-trihydroxyacetophenone. For example, resolution for 5-12-mer oligonucleotides is greater than 7000 using overlayer matrix preparations and mass accuracy values are well below 20 ppm. In addition to these methods, a new method for analyzing DNA in positive ion mode is reported using acidified 3-hydroxypicolinic acid. This method does not lose sensitivity for higher mass oligonucleotides as quickly as overlayer methods, and spectra retain > 6000 resolution and mass accuracies of approximately 20 ppm between different overlayer depositions.

DNA↗

PubMed2000

Two-dimensional fluorescence correlation in capillary electrophoresis for peak resolution and species identification.

A new spectroscopic dimension-fluorescence intensity correlation--is introduced to enhance peak resolution and species identification in capillary electrophoresis. In two-dimensional correlation CE, a conventional electropherogram is spread into two dimensions through cross-correlation analysis of fluorescence time response. A laser that is sinusoidally modulated in intensity is used as the excitation source. Three channels of information are collected during a CE run: the steady-state intensity, the ac amplitude, and the phase-resolved fluorescence intensity. The correlation between two chosen channels is then evaluated. A two-dimensional correlation electropherogram consists of a plot of the correlation intensity versus two axes of migration time. Through correlation analysis, species discrimination and peak resolution are significantly enhanced without having to physically separate the solutes. Two-dimensional correlation CE showed complete resolution between two overlapping sample peaks with a resolution of 0.28 in the conventional one-dimensional electropherogram. In separations of polycyclic aromatic hydrocarbons by micellar electrokinetic chromatography (MEKC), two-dimensional correlation analysis resolved all overlapping elution peaks unseparable by one-dimensional MEKC, demonstrating the utility of 2D correlation in separation method development. The capability of 2D correlation CE in species identification is demonstrated with a sequence of 39 consecutively injected peaks containing four fluorescent dyes. Species identification in sequencing is achieved without complex data treatment in two-dimensional correlation CE.

Electrophoresis, Capillary↗

PubMed2005

Resolution of small molecules by passage through an open capillary.

When a solute passes through an open capillary, in which a laminar flow is established, different peak profiles can be obtained according to its diffusion property under a working condition, i.e., the radius and length of the capillary and the flow rate of the carrier solution. If a solute diffuses over the entire cross section of the capillary before it is eluted, a Gaussian-shaped diffusion peak appears, which has an apex at the travel time of the average flow. Insufficient solute diffusion, which is realized, e.g., by increasing flow rates or capillary radius, produces a new peak having an apex at the travel time of the maximum flow. This implies that two solutes can be resolved simply by passing through a capillary. However, our previous study indicated that the diffusion coefficients of two solutes should be at least one order different for their resolution based on this principle, suggesting that its applicability is highly restricted. In the present paper, this concept has been extended to the resolution between dissolved solutes that have similar intrinsic diffusion properties. The incorporation of molecular aggregates in the carrier makes a solute less diffusive according to the extent of their interaction and allows the resolution of a dissolved molecule from other ones differing in the affinity to the molecular aggregates. Several examples of peak resolution for phenols, aromatic hydrocarbons, and inorganic anions are shown and discussed on the basis of the modification of the diffusion natures due to their interactions with micelles or vesicles.

Journal Article↗

PubMed2006

Monitoring and modeling of protein processes using mass spectrometry, circular dichroism, and multivariate curve resolution methods.

Mass spectrometry has recently become one of the major analytical tools to study biomolecular structure and function. Ionization techniques, such as electrospray ionization (ESI), desorb biomolecules from solution to the gas phase keeping practically intact their natural structure. ESI applied to a protein solution produces a mixture of multiply charged ions, the ion charge distribution of which depends on the oligomeric form (mass) and on the protein surface exposed (amount of accommodated charges) of the related protein conformation. ESI-MS provides an efficient way to monitor protein processes; however, the ionic contributions of the different protein conformations involved usually overlap, and the use of chemometric tools is necessary to unravel the information related to the pure conformations that the biomolecule adopts along the process. Multivariate curve resolution-alternating least squares applied to MS-monitored protein processes provides the concentration profiles associated with the different protein conformations occurring during the process and the related pure mass spectra. The concentration profiles, in this context, the ionic contributions, describe the process mechanism and the structural information derived from the pure mass spectra characterizes the involved conformations. Mass spectra can be expressed schematically through percentages of base peak intensity. This chemical transformation compresses significantly the raw spectra and allows for an easier application of natural MS-related constraints, such as the presence of only one maximum, i.e., the base peak of a particular conformation, into the resolution of the pure signals. The combination of mass spectrometry and multivariate curve resolution methods is used to elucidate the mechanism of the pH-induced conformation changes of the bovine beta-lactoglobulin. As a final step, MS data are fused with circular dichroism data and are simultaneously analyzed to ensure and confirm that all the previously detected MS conformations really exist in solution and are an artifact of neither the ionization process nor their chemometric resolution.

Animals↗

PubMed1996

Exact mass measurement of polar organic molecules at low resolution using electrospray ionization and a quadrupole mass spectrometer.

Hitherto, exact mass measurement experiments have usually been performed using high-resolution mass spectrometry. However, under the right circumstances, measurements with comparable accuracy may be made at low resolution. Here we demonstrate the use of a low-resolution single-quadrupole mass spectrometer to accurately mass measure organic samples analyzed by electrospray ionization, using a variety of glycol polymers for internal calibration. Results are presented from 11 samples which yield molecular signals in the m/z range 190-750, including data for positive, negative, and multiply-charged sample and reference ions. Replicate determinations of the masses of 12 ions gave values within 4.5 ppm (1.1 millimass units, mmu) of their calculated values, with standard deviations no larger than 3 ppm (1.7 mmu). From a total of 88 individual 1 min measurements, 83 were within 5 ppm, and 87 within 2 mmu of the theoretical mass. The accuracy, precision, and sensitivity shown here are comparable to those achievable using high-resolution mass spectrometers, with the added benefits of simpler instrumentation and analytical technique afforded by the quadrupole mass analyzer.

Bradykinin↗