Biology Commons^™

Cathodoluminescence Image Processing Of High Tc Superconductors, Z. Barkay, G. Deutscher, E. Grunbaum

Scanning Microscopy

Cathodoluminescence (CL) in the Scanning Electron Microscope (SEM) was performed for both ceramic pellets and thin films of YBaCuO high T_C superconductors. Image processing provided additional quantitative information. For single phase films, we demonstrated the possibility to create thickness maps in real time from the CL pictures. The gradual thickness variation within the sample was revealed by the histogram of the thickness image. The continuity of the film was observed at a few threshold thicknesses values, defined by the fraction of the occupied area. At the conduction threshold value, the location and width of the conducting paths could be …

Direct Methods In High Resolution Electron Microscopy, D. Van Dyck, M. Op De Beeck

Scanning Microscopy

New approaches are proposed to retrieve the wavefunction at the object and from this, to retrieve the projected structure of the object. The wavefunction is retrieved by capturing images at a series of closely spaced focus values and to process the whole 3D data. The structure of the object is retrieved using a formalism based on electron channelling.

Morphological Three-Dimensional Analysis, C. Gratin, F. Meyer

Scanning Microscopy

This paper presents the main results of our research on mathematical morphology for three-dimensional images. The first issue is to decide which grid must be used. The face-centred cubic grid and the centred cubic grid seem to be more suitable than the cubic grid in terms of possible rotations. For these two grids we derive the formulae for the basic Minkovski measures. Then we show through several examples that extension from 2D to 3D is straightforward for most transformations. The efficiency of direct 3D processing is illustrated by applications to filtering, overlapping particle separation and grey-scale image segmentation.

An Optical Moiré Technique For The Analysis Of Displacements In Lattice Images, C. J. D. Hetherington, U. Dahmen

Scanning Microscopy

High resolution electron microscopy is a means for imaging not only the local structure at a defect or interface but also the displacement field in the surrounding lattice. However, in general it is difficult or tedious to analyse this field which can extend across the entire micrograph. The optical moiré technique, which is based on interference effects between the experimental lattice image and an artificial reference lattice, allows a rapid and accurate measurement of the displacements. Small violations of translation, rotation or mirror symmetries give rise to large changes in the periodicity or orientation of the moiré pattern.

The spacing …

Application Of Slow-Scan Charge-Coupled Device (Ccd) Cameras To On-Line Microscope Control, O. L. Krivanek, G. Y. Fan

Scanning Microscopy

Autotuning methods for transmission electron microscopy are reviewed, and a distinction is drawn between predictive and non-predictive methods. The predictive methods make better use of the input data and therefore need fewer images to carry out complete autotuning. They typically require high quality of input data, which can be best provided by cooled slow-scan charge-coupled device (CCD) cameras. Two predictive methods are considered in more detail. These are the tilt-induced image shift (TIS) method of Koster, van der Mast and de Ruijter, and a new automated diffractogram analysis (ADA) method, which is introduced in this paper. The ADA method is …

Measurement Of Electron-Optical Parameters For High-Resolution Electron Microscopy Image Interpretation, A. F. De Jong, A. J. Koster

Scanning Microscopy

A method is presented to measure various electron-optical parameters needed for high-resolution electron microscopy image interpretation with high accuracy. The method is based on the measurement of a series of beam-tilt induced image displacements. The displacements are calculated via cross-correlation of the images, and subsequently fitted to a third-order polynomal in the beam tilt. From two series of images (using the x and y beam tilt coils), the spherical aberration constant of the microscope can be measured, as well as the current values of defocus, beam tilt and astigmatism. The spherical aberration constant of three Philips microscopes is measured with …

Imaging Deformed Proteins: Characterising The State Fully, W. O. Saxton

Scanning Microscopy

By analysing the positions of individual unit cells in the image of a distorted macromolecular crystal, it is possible to achieve considerably more than is achieved by the correlation averaging or unbending now widely practised. It is possible partly to compensate for individual molecular distortion; and it is possible to identify molecules in equivalent environments (which can be expected to be in equivalent states of strain), selective averaging of which yields images that show how strain is accommodated at the sub-molecular level. The possible presence of surface forces applied to the crystal by its support film complicates the analysis and …

The Watershed Transformation Applied To Image Segmentation, Serge Beucher

Scanning Microscopy

Image segmentation by mathematical morphology is a methodology based upon the notions of watershed and homotopy modification.This paper aims at introducing this methodology through various examples of segmentation in materials sciences, electron microscopy and scene analysis.

First, we defined our basic tool, the watershed transform. We showed that this transformation can be built by implementing a flooding process on a greytone image. This flooding process can be performed by using elementary morphological operations such as geodesic skeleton and reconstruction. Other algorithms are also briefly presented (arrows representation).

Then, the use of this transformation for image segmentation purposes is discussed. The …

Phase Space Methods In Image Formation Theory, Victor Castaño, Gustavo Vásquez-Polo, Ramón Gutiérrez-Castrejón

Scanning Microscopy

A review of the basic mathematical principles and properties of some phase space functions (Wigner and Ambiguity functions) is first presented in a more general mathematical context. Then, we deal in detail with three applications : image formation in optical systems (specifically cascading systems of lenses), image formation in Transmission Electron Microscopy, by taking advantage of some formal similarities with the previous examples and, finally, contrast transfer functions in Electron Microscopy in order to obtain 2-dimensional plots which provide information on the behavior of a transmission electron microscope. Also, the relations of these functions with other mathematical tools utilized in …

Random Image Models For Microstructure Analysis And Simulation, Dominique Jeulin

Scanning Microscopy

A wide spectrum of Random Function models, based on the theory of Random sets, are introduced to simulate single or multivariate signals as encountered in the common practice of electron probe microscopy.

These models are built in three steps, combining the choice of a family of primary random functions and of Poisson varieties in the n-dimensional space for their implantation. For electron microscopy images, they can describe the following situations:

- topography (as obtained from stereo pair images in fractography) simulated by Boolean and by alternate sequential random functions;

- thick slices (as in the case of Transmission Electron Microscope …

Some Applications Of Image Analysis To Materials Science, François Grillon

Scanning Microscopy

We have presented four applications of Image Analysis to material science. The first one is on a yarn composite SiC where we want to know the relative proportion of matrix, fibers, porosity and the size of the fibers. The results are used to estimate mechanical properties of the composite.

The second one is to measure the residual porosity after a laser shock on powder metallurgy steel. The knowledge of the depth of the affected zone is necessary to optimise the laser treatment.

The third one uses Fourier transformation to analyse plastic deformation on grains. Values are obtained from Fourier transform …

Diffraction And Imaging Theory Of Inelastically Scattered Electrons - A New Approach, Z. L. Wang, J. Bentley

Scanning Microscopy

A new dynamical theory is developed for describing inelastic electron scattering in thin crystals. Compared to existing theories, the first advantage of this new theory is that the incoherent summation of the diffracted intensities contributed by electrons after exciting vast numbers of degenerate excited states has been evaluated before any numerical calculation. The second advantage is that only the modulus squared of the transition matrix elements are needed in the final computation. This greatly reduces the effort in searching for "phase shifts" in inelastic scattering matrix calculations. By iterative operation of this single-inelastic scattering theory, multiple-inelastic electron scattering of phonons, …

The Application Of Multispectral Techniques To Analytical Electron Microscopy, P. G. Kenny, M. Prutton, R. H. Roberts, I. R. Barkshire, J. C. Greenwood, M. J. Hadley, S. P. Tear

Scanning Microscopy

The York multispectral analytical electron microscope (MULSAM) was the first instrument specifically designed to acquire and process multiple Auger, X-ray, backscattered electron, elastically scattered electron, scanning electron microscopy (SEM) and specimen absorption current images simultaneously. Analyzing combinations of these signals with multispectral correlation techniques yields more information than would normally be obtained by treating each image separately. This paper reports some of the multispectral methods we have investigated at York which may be of use to other workers. Included are (1) a method that corrects for beam current fluctuations during long acquisition runs which is based on the anti-correlation between …

A Brief Introduction To Aims And Methods For Image Processing Of Electron Micrographs Of Biological Specimens, Murray Stewart

Scanning Microscopy

A common problem with electron micrographs of biological objects is that fine details are usually faint and, moreover, tend to be obscured by background noise from stain and support film. Filtering is a useful way to improve the signal-to-noise ratio and is particularly important when trying to detect small changes (conformational or due to labelling) or when examining frozen hydrated objects, where the statistical definition of the image has been reduced because of the low-dose conditions needed to prevent radiation damage. Filtering can also be used to address the superposition effects that result from the large depth of focus of …

Three-Dimensional Crystallographic Reconstruction For Atomic Resolution, Kenneth H. Downing

Scanning Microscopy

Three-dimensional structures have recently been determined by electron crystallography at a resolution high enough to determine atomic arrangements in both protein and mineral specimens. The different nature of these two types of specimens produces some very significant differences in the way data is obtained and processed, although the principles are the same. The sensitivity of proteins to damage by the electron beam limits the signal-to-noise ratio in the image and the resolution to which data can be extracted from the image. A number of constraints, such as the amino acid sequence and the connectivity of atoms within amino acids, can …

Alignment, Classification, And Three-Dimensional Reconstruction Of Single Particles Embedded In Ice, Joachim Frank, Pawel Penczek, Weiping Liu

Scanning Microscopy

Cryo-electron microscopy of single biological particles poses new challenges to digital image processing due to the low signal-to-noise ratio of the data. New tools have been devised to deal with important aspects of 3-D reconstruction following the random-conical data collection scheme: (a) a new shift-invariant function has been derived, which promises to facilitate alignment and classification of single particle projections; (b) a new method of orientation search is proposed, which makes it possible to relate random-conical data sets to one another prior to reconstruction; and (c) the foundation is laid for a 3-D variance estimation which utilizes the oversampling of …

Confocal Microscopy And Three-Dimensional Reconstruction Of Thick, Transparent, Vital Tissue, Barry R. Masters

Scanning Microscopy

The three-dimensional visualization of the 400 micron thick, transparent, in situ cornea is described to demonstrate the use of confocal light microscopy for noninvasive imaging of living cells and thick tissues in their normal, vital conditions. Specimen preparation and physiological stability, as well as light attenuation corrections are critical to data acquisition. The technique to provide mechanical stability of the specimen during the duration of the image acquisition is explained. A laser scanning confocal light microscope (LSCM) was used to obtain optical serial sections from rabbit eyes that were freshly removed and placed in a physiological Ringer's solution. This study …

Structure Analysis Of Ice-Embedded Single Particles, M. Van Heel, H. Winkler, E. Orlova, M. Schatz

Scanning Microscopy

The conventional negative-stain preparation method for electron microscopy, in which biological macromolecules are contrasted using heavy metal salts (such as uranyl-acetate), is a simple and fast technique which has helped visualize hundreds of different molecular structures. Computer analysis of such negatively stained images of individual (i.e., non-crystalline) macromolecules using statistical pattern-recognition techniques has revealed considerable new structural information. Negative staining, however, has some disadvantages: the specimens are often severely flattened (as much as 25%-75%), they often exhibit strong preferential attachment of the molecules to the supporting carbon foil, and the molecular images may be difficult to interpret due to the …

Morphological Filtering And Granulometric Analysis On Scanning Electron Micrographs: Applications In Materials Science, Murielle Prod'homme, Michel Coster, Liliane Chermant, Jean-Louis Chermant

Scanning Microscopy

For many applications, scanning electron microscopy (SEM) images reflect the granular texture of analysed objects. So it is important to characterise the morphology of this texture and also to filter these images. Because the size of the texture is the main criterion to be studied, we have focused our paper on granulometric analysis.

We present basic parameters, morphological filtering and granulometry for Rⁿ•R functions and their properties with local knowledge and anamorphosis.

Some applications in the domain of materials science illustrate these methods and present their suitable possibilities.

Application Of The Wavelet Transform To The Digital Image Processing Of Electron Micrographs And Of Backreflection Electron Diffraction Patterns, A. Gómez, L. Beltrán Del Río, D. Romeu, M. Jose Yacamán

Scanning Microscopy

In this work we explore the use of the so-called wavelet transform in the digital image processing of micrographs. The wavelet transform of an image f(x,y) is defined as:

Wf(s,u,v)

= f(x,y) s Ψ(s(x-u),s(y-v)) dxdy

where Ψ is an analyzing function called "wavelet" and which is in our examples always taken to be the "Mexican hat" given by

Ψ(x)=(2-(x²+y²))exp(-(x²+y²)/2)

Some synthetic images are shown in which it can be clearly seen how the wavelet transform can be useful to reveal edges and to emphasize the boundaries of the clusters.

The technique is …

Image Algebra And Restoration, P. W. Hawkes

Scanning Microscopy

The arrival of image algebras has made it possible to express a vast amount of the heterogeneous material in the image processing literature in a convenient and consistent manner. Furthermore, this type of formulation has proved very fertile and many new ideas have emerged. Image restoration has, however, been studied very much less than enhancement and analysis. We explain briefly the use of one such algebra in this field and show that such tasks as reconstruction from focal series and three-dimensional reconstruction can easily be incorporated.

A Robust Solution To The Super-Resolution Phase Problem In Scanning Transmission Electron Microscopy, J. M. Rodenburg, B. C. Mccallum

Scanning Microscopy

From a set of images, each of poor resolution, recorded in a transmission microscope under many different incident angles of coherent illumination, it is possible to obtain wavelength-limited resolution even if there is a narrow aperture lying in the back-focal plane of the imaging lens. This is achieved by a deconvolution algorithm which retrieves the phase of the Fourier transform of the specimen. The method accounts for complex components in the transfer function of the lens, is not very sensitive to defocus and is remarkably resilient to noise. It may have important applications in overcoming the resolution limit in the …

The Detection And Quantification Of Straight-Lined Irregularities On Surfaces, J. Alan Swift

Scanning Microscopy

Under the microscope, scratches or abrasions on hard otherwise flat surfaces are usually revealed as straight-lined irregularities. At a more macroscopic level creases in thin sheets such as of paper and textile fabrics are also observed to be straight-lined. A computer-aided image analytical method is described here not only for identifying such features but also for counting them, measuring their lengths and evaluating their contrast. Further measures are derived that are in accord with the qualitative visual impact of each line within the milleau of lines in the original image. The method makes use of a parametric transformation from two …

Multi-Dimensional Data Analysis And Processing In Electron-Induced Microanalysis, N. Bonnet, P. Trebbia

Scanning Microscopy

The new facilities offered by computer controlled data capturing devices allow one to open the field of data acquisition from one or two-dimensional spaces to multi-dimensional ones. The methods used for analysing and processing such data sets have to move in parallel towards multi-dimensionality. Multivariate Statistical Analysis is one of the tools which appear to be promising in : data analysis, data reduction, data processing (multivariate noise filtering), data interpolation and extrapolation. Illustrations of these different possibilities are given in the fields of spatially resolved spectroscopy, time-dependent spectroscopy and elemental mapping from Electron Energy Loss Spectroscopy.

Processing Multi-Spectral Scanning Electron Microscopy Images For Quantitative Microfabric Analysis, N. K. Tovey, D. L. Dent, W. M. Corbett, D. H. Krinsley

Scanning Microscopy

Multi-spectral image analysis is a powerful method to characterise quantitatively the mineralogy and microfabric of soils, sediments, and other particulate materials. Backscattered scanning electron microscope (SEM) images of polished, resin-impregnated samples are grouped with the corresponding X-ray elemental maps using classification methods commonly used in remote sensing. However, the resulting mineral-segmented images require processing to render them suitable for quantification. In the past, this has been done subjectively and interactively, but the new objective methods described in this paper largely eliminate this subjectivity. An intensity gradient magnitude image of the original backscattered electron image is used as the basis of …

Image Segmentation Applied To Scanning Electron Microscopy Multi-Images Of Weathered Stones Of Monuments, M. Rautureau, R. Harba, G. Jacquet

Scanning Microscopy

This paper describes a three complementary images processing method. The three images are coming from a scanning electron microscope (SEM) during the analysis of a particular stone: the Tuffeau used in most monuments of the Loire valley (France). The goal is to separate two classes of particles (calcareous and siliceous) from the porosity to give more information to experts who evaluate the damage of weathering on monuments. A specific process is developed: a first threshold on the good quality image allows separation of particles from porosity. Then, the complementarity of the three images gives the two other thresholds. Granulometry, percentages …

Random Access Direct Parallel Detection Of Electron Energy Loss Spectra With A New Photodiode Array, Stephan La Barré, Eckhard Quandt, Heinz Niedrig

Scanning Microscopy

A new type of photodiode array (PDA) which allows the individual access of single diodes is used to record energy loss electrons directly without electron photon conversion.

The detector is part of an electron energy loss parallel detection system which consists of a sector field spectrometer and a quadrupole lens system to magnify the spectra electron-optically.

The parallel detector is a new random access PDA (RAPDA) which allows the individual access of single diodes. Therefore, different parts of the spectrum with dramatically different electron rates (zero loss peak, plasmon loss, ionization edges) can be recorded, each with optimum acquisition time …

On-Line Processing And Computer Control In High Resolution Transmission Electron Microscopy, F. A. Ponce, H. Hikashi

Scanning Microscopy

Recent advances in hardware and software make it possible to perform real-time image processing, and to do simultaneous analysis of the image characteristics in the transmission electron microscope (TEM). We have used a commercially available image processor and a suitable computer/TEM interface to build a automated computer-control system for a JEOL 4000EX TEM. We report here the characteristics of the system, which has direct access to all electronic controls of a JEOL 4000EX. The various microscope parameters are set by changing one parameter at a time while the image processor measures a specific image variable such as the variance. From …

Quantification Of High-Resolution Lattice Images And Electron Holograms, W. J. De Ruijter, M. Gajdardziska-Josifovska, M. R. Mccartney, R. Sharma, David J. Smith, J. K. Weiss

Scanning Microscopy

Progress towards the quantification of high-resolution electron microscopy and electron holograms has been achieved using digital acquisition with a slow-scan charge-coupled device (CCD) camera. Two applications are described: the precise measurement of lattice-fringe spacings and the determination of the mean inner potential. Lattice images can be characterized by a finite sum of two-dimensional sinusoids. A new method for measurement of the frequency, amplitude and phase of each sinusoid, based on an interpolation technique in reciprocal space, is presented. The method offers considerably higher precision for measurement of lattice fringes than the optical bench and is applicable to images recorded with …

Digital Image Processing Of Electron Micrographs: The Pic System Ii, Benes L. Trus, Michael Unser, Thierry Pun, Alasdair C. Steven

Scanning Microscopy

The PIC system, an integrated package of Fortran programs and subroutines designed to run on the Digital Equipment Corporation VAX family of computers, has been developed for analysis of electron micrographs with emphasis on the particular requirements for structural analysis of biological macromolecules. The substantially improved VAX version of PIC reported here has been developed from an earlier PDP-11 version which was, in turn, developed from a set of IBM 370 programs called MDPP. PIC now encompasses over 150 commands or processing operations that afford a comprehensive range of image processing operations including image restoration, enhancement, Fourier analysis, correlation averaging, …