Biomedical subjects

M Unser

Publications and source records attributed to M Unser.

13 recordsLinked to original sources

PubMed1992

Magnification mismatches between micrographs: corrective procedures and implications for structural analysis.

Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.

Algorithms↗

PubMed1992

Automated detection of the tongue surface in sequences of ultrasound images.

An image processing system has been developed for a Macintosh II personal computer. It is designed to process sequences of sagittal tongue sections that are digitized in real time and stored in standard tagged image file format (TIFF). The successive processing steps are: (a) a low-pass filter for noise reduction, (b) a resampling of the sector of interest in polar coordinates, (c) a matched filter (vertical differentiator) for the enhancement of the tissue/air interface in the surface region of the tongue, and (d) an extraction of border points by searching for an optimal radial path along the angular dimension. This latter task is achieved by dynamic programming, which has the following advantages. First, due to the use of a global criterion to guide the detection, it is very robust. Second, as a result of certain restrictions of the allowable transitions, the extracted contours are smooth. Finally, the method permits the specification of particular predefined contour points. This system was implemented in a program that can handle image sequences in a fully automatic mode. Results obtained using ultrasound data are presented.

Humans↗

PubMed1991

Computerized methods for analyzing two-dimensional agarose gel electropherograms.

Previous methods interpret zonal or polydisperse gel patterns of two-dimensional Serwer-type gels in terms of size and free mobility (surface net charge density). These two parameters have been determined for each component without quantitatively measuring the abundance of the components. The present study advances these previous methods by determining the relative concentration of each component by computer evaluation of densitometrically analyzed gel patterns. Suitable procedures and their underlying algorithms are presented. The mathematical routines are implemented in a user-friendly software package, called GelFit and designed for a Macintosh personal computer. The program input consists of digitized images of gel staining patterns exemplified by those obtained from electrophoresis of native subcellular-sized particles. The data are processed through the following steps: (i) Noise reduction and calibration. (ii) Geometrical transformation of the pattern onto a rectangular size/free mobility coordinate system using rationales of the extended Ogston model. (iii) Analysis of the transformed image to determine density maxima, density profiles along iso-free-mobility or iso-size lines, curve fitting of one-dimensional profiles or two-dimensional surfaces using Gaussian functions and curve stripping of surfaces to determine the possible number of particle populations.

Algorithms↗

PubMed1991

The distribution of particles characterized by size and free mobility within polydisperse populations of protein-polysaccharide conjugates, determined from two-dimensional agarose electropherograms.

New approaches for the characterization of polydisperse particle populations are presented*. The investigated samples contain virus-sized protein-polysaccharide conjugates which had previously been prepared as immunogens against bacterial meningitis (Hib). The analysis is based on two-dimensional agarose electrophoresis (Serwer-type). This method, like the one of O'Farrell, achieves a separation according to size and charge. It relies on a different principle, however, and is applicable to nondenatured particles which are 100 to more than 1000 times larger in mass than regular uncrosslinked proteins. Data from stained gel patterns are evaluated by the computer program ELPHOFIT, which makes it possible to standardize the gel and to construct a nomogram which defines every position on the gel in terms of particle size and free mobility (related to surface net charge density). The output of ELPHOFIT, consisting of nomogram parameters, is transferred to the image processing program GELFIT. This software is used to evaluate the computer images obtained by digitizing the stained gel patterns: (i) The nomogram is electronically superimposed on the computer image. (ii) The gel pattern is transformed from a curvilinear to a rectangular coordinate system of particle size and free mobility. The center of gravity as well as density maxima are given in coordinates of particle size and free mobility. Ranges of grey levels can be accentuated by adding 16 pseudocolors. (iii) Using surface-stripping techniques, GELFIT provides an estimate for the number of major subpopulations within each preparation. (iv) Numerical values for the distribution of particle size and free mobility are determined. Using program IMAGE, the quantitative physical assessment of a given conjugate preparation is presented in the form of a computer-generated three-dimensional plot, the shape of which serves to identify and characterize the preparation visually. The data analysis based on digitized two-dimensional gel patterns is automated to an extent that a technician can perform routine evaluations. It uses the Macintosh II personal computer.

Bacterial Outer Membrane Proteins↗

PubMed1991

Spatial disorders and computational cures.

Image averaging provides a powerful method for enhancing the yield of interpretable information from electron micrographs of biological macromolecules. However, as originally conceived, the full benefit of averaging is achieved only with perfectly ordered two-dimensional crystals. More recent developments, reviewed here, allow one to rectify disordered lattices, straighten randomly bent filaments, and combine multiple images of free-standing particles, thus extending the advantages of image averaging to virtually every class of macromolecular specimen.

Animals↗

PubMed1990

Computerized cataract detection and classification.

156 eyes of patients and normal volunteers were classified at the slit lamp into the following pure groups: normal (n = 50), nuclear (n = 39), cortical (n = 33) and PSC (n = 34). The eyes were photographed with the Topcon SL-45 Scheimpflug camera and the images scanned and processed to obtain one dimensional profiles through a 40 x 440 micron axial window. Of the 156 profiles, 90 were used as reference samples and were processed to obtain average profiles. The remaining 66 "unknown" profiles and each of the reference profiles, were classified into the four groups based on their distance from the average profiles in Euclidean space. The system was found to be very sensitive (98%) in detecting the presence of cataracts and specific (100%) in identifying normal, i.e. cataract negative lenses. In classifying pure cataracts into the various classes 98% of answers were correct.

Adult↗

PubMed1989

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.

Actins↗

PubMed1988

Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7.

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.

Amino Acid Sequence↗

PubMed1987

A new resolution criterion based on spectral signal-to-noise ratios.

A new criterion for the "useful" resolution of electron micrographs of macromolecular particles is introduced. This criterion is based on estimation of the spatial frequency limit beyond which the spectral signal-to-noise ratio (SSNR) falls below an acceptable baseline. Applicable to both periodic and aperiodic specimens, this approach is particularly apposite for sets of correlation-averaged images. It represents a straightforward and intuitively appealing generalization of the traditional method of estimating the resolution of crystalline specimens from the spectral ranges of periodic reflections in their diffraction patterns. This method allows one to assess how closely the resolution of an averaged image based on N individual images approaches the ultimate resolution obtainable from an indefinitely large number of statistically equivalent images. Inter-relationships between the SSNR and two other measures of resolution, the differential phase residual and the Fourier ring correlation coefficient, are discussed, and their properties compared.

Biopolymers↗

PubMed1987

Optimal background estimation in EELS.

In quantitative electron energy loss spectrometry, it is desirable to estimate the background law below core edge energy in a way that provides the maximum signal-to-noise ratio. Assuming an inverse power background model and independently Poisson distributed measurements, it is shown how to achieve this goal by using a maximum likelihood (ML) estimation technique which provides unbiased and minimum mean square error estimates of all parameters of interest. An efficient and computationally stable implementation of this procedure is proposed. Standard logarithmic least squares estimations are then compared with the ML approach and the gain in performance due to optimal processing is quantified.

Electrons↗

PubMed1986

Odd men out: a quantitative objective procedure for identifying anomalous members of a set of noisy images of ostensibly identical specimens.

Achievement of an optimal improvement in signal-to-noise ratio from image averaging techniques depends crucially on the assumption that all members of the set of images to be averaged are fundamentally alike. In HREM of biological macromolecules, this assumption may be invalid for such reasons as variations in viewing geometry, non-uniformity of staining, or structural perturbations caused by specimen preparation procedures of radiation damage. Inclusion of data that are compromized these or other factors will degrade the information content of the averaged image. Here we present an algorithm which provides an objective quantitative method for the identification and elimination of anomalous members of a set of pre-aligned images. Based on a statistical criterion of mutual consistency, the algorithm forms an ordered list in which the individual images are ranked from most to least reliable. On specification of the noise statistics--in the formulation given here, of stationary white noise--an acceptability threshold in this ordered list is imposed. The derivation and implementation of this algorithm are presented, its properties discussed, and its application illustrated using both real and model electron micrograph data.

Algorithms↗

PubMedDate not supplied

Normalization procedures and factorial representations for classification of correlation-aligned images: a comparative study.

We have addressed the problem of optimizing procedures of multivariate statistical analysis (MSA) for identifying homogeneous sets of electron micrographs of biological macromolecules, with a view to averaging over consistent sets of images. Using pre-aligned images of negatively stained protein molecules - known a priori to fall into two subtly different classes - we compared how the capacity to discriminate between them was affected by the normalization procedure used, and by the choice of factorial representation. Specifically, these images were analyzed both after being scaled according to constant minimum and maximum (CMM) values, and after imposing constant values of image mean and variance (CMV). The factorial representations compared were correspondence analysis (CA) and the principal components (PC) formalism. When used with PC, CMM normalization was found to give rise to spurious inter-image fluctuations that were more pronounced than the genuine difference between the two kinds of images; even with CA, CMV proved to be a more satisfactory method of normalization. When CMV was used with CA or PC, both factorial representations yielded qualitatively similar results, although according to a quantitative measure of inter-set discrimination, the performance of PC was slightly superior. Even in the best case, however, the two classes of images - as mapped in factorial space - were not fully resolved. The implications of this observation are discussed with regard to potential ambiguities of image classification in practice.

Classification↗

PubMedDate not supplied

The spectral signal-to-noise ratio resolution criterion: computational efficiency and statistical precision.

This note describes a practical improvement in the computational efficiency of the spectral signal-to-noise ratio (SSNR) resolution criterion for correlation-averaged images. The total set of N images is randomly partitioned into ng subsets, each subset is separately averaged, and a reduced form of the SSNR is computed from these average images. In general, larger values of ng achieve lower statistical uncertainty, while smaller values of ng are computationally more expedient. It is shown that, for negatively stained data, a judicious compromise is achieved with 10 less than or equal to ng less than or equal to 20, regardless of how large N may be.

Microscopy, Interference↗