cabios communication - Semantic Scholar

CABIOS COMMUNICATION

Vol. 12 no. 3 1996 Pages 241-245

IBIS version 3: An OSFI Motif-based interface for IBIS—integrated biological imaging system Jean-Paul RollancT, Mohamed J.FIifla1, Mireille Garreau and Daniel Thomas Abstract

Introduction Visualization of biological matter in electron microscopy at a resolution ranging 0.3-3.0 nm needs to extract the full information content from the primary electron microscopic data, using image processing. To fulfill these needs, several softwares have been developed (Smith, 1978; Saxton el al, 1979; Frank et al., 1981; Trus and Steven, 1981; Van Heel and Keegstra, 1981; Hegerl and Altbauer, 1982; Crowther et al., 1996). The first IBIS version has been presented in 1992 (Flifla et al., 1992). This software was developed essentially for structural analysis of biological macromolecules (Thomas et al., 1988). Although the original program was flexible, as a result of its modular conception, it was necessary to use an external display software for visualizing the results of a given operation. X-windows is a flexible graphical environment highly portable on several systems. Many programs which need graphical or image facilities are now running under Xwindows. On a window-based presentation, IBIS v.3 provides all the procedures from the original version with the addition of new modules. The program is organized in a very interactive and user-friendly manner with a number Biologie Cellulaire el Reproduction, URA CNRS no. 256 Universite de Renncs 1, Campus de Beaulieu. 35042 Rennes and' Ecole Louis de Broglie, 35170 Bruz, France * To whom correspondence should be addressed. E-mail: [email protected]

© Oxford University Press

System and methods IBIS version 3 is developed with Motif 1.2, a toolkit from the Open Software Foundation (OSF) written for the Xwindow system. Most of the mathematical functions are written in FORTRAN 77, while C is used for the part developed under Motif-based windows and also for the new integrated calculation sequences included in IBIS v.3. Data organization IBIS version 3 data organization is presented in Figure 1. RAM The memory is divided into five parts. The first one is composed of two temporary elements named Tempo-E and Tempo-S. Each array element is coded as an eight byte word. These arrays are used for high precision computing. The second part of the memory is made of a set of 12 elements called BETA. BETA is written on one, two, or four bytes according to the available memory space of the system used. The use of such arrays avoids multiple access to disk files and thus saves time. When sub-images are extracted from original images, they are stored on the first eight arrays of BETA. During the process of sub-images, all the computations are achieved using BETA, Tempo-E, and Tempo-S memories. After processing, only the first eight arrays of BETA are saved simultaneously, however saving sub-images independently is also possible. The third part of the memory is used for the quasi optical diffraction function. The fourth part is used for interpolation, and the last part is used for windowing sequences. Disk files Images are stored as one byte pixel arrays. Sub-images extracted from raw images can be stored directly on disk files and are called ALPHA in IBIS system. This ALPHA file is composed of eight elements of 128 x 128 pixels each and quoted from one to eight. One sub-image is one element in this ALPHA file. When an ALPHA file is read

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IBIS is a set of computer programs dedicated to the processing of electron micrographs, mainly for structural analysis of biological macromolecules. We present IBIS version 3, a UNIX/OSF/Motif 1.2-based package which carries out and provides visual display of the many operations essential to image analysis. To ensure portability, the software is written in FORTRAN 77 for computing mathematical functions and in C for display routines. A description of the IBIS OSF I Motif interface is given with the new functions added to the original version. IBIS v.3 is available free of charge to other laboratories on the internet via anonymous ftp (URL: ftp:/'/J~tp.univ-rennesl.fr7'pub/ incoming/'1'BIS. tar.Z).

of functions accessible from pull-down menus. Each one gives access to a specific environment with its own input/ output.

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OOD Fig. 1. Data organization of IBIS v.3. Subimages are extracted from a raw image, then stored on the first eight elements of BETA. For the processing of subimages, the computation is achieved using BETA, Tempo-E, and Tempo-S. Then images are stored on disk as an eight-element ALPHA file. SIGMA is an automatic backup of BETA, used to recover lost data Quasi optical diffraction (QOD) and interpolation (IP) procedures use two independent matrices of 512 x 512 pixels.

from the disk, its eight elements are stored on thefirsteight arrays of BETA. A backup of BETA is automatically stored on disk file (SIGMA) after each calculation sequence and can be used to recover lost data in case of system interruptions. The Motif interface As shown in Figure 2, the main window of IBIS v.3 is divided into five parts, namely: a display area, a loading area, a general information area, a menu area, and an image display adjusting area. A temporary external drawing area is activated only for windowing procedures. Images with a maximum size of 512 x 512 pixels are displayed on the left-hand side drawing area of the main

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window. The bottom part of the drawing area is used only to display gray level histograms. On the upper right-hand side is the loading push-button area and the general information area. From this area one can access to a file selection box and to various functions such as thresholding, image statistics, gray level histogram, intensity profile lines and quasi optical diffraction. The information area contains the current file name and two text areas which are respectively a session history which is saved in a file, and a multipurpose area for help messages, step by step instructions, pixel co-ordinates and intensity, output and error messages. The right-hand side contains the image processing menu in which each function is associated with a cascadebutton. These widgets allow easy choice of a given


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Fig. 2. IBIS main window developed in this work. The display area shows 12 subimages of isolated half-molecules of a-2-macroglobulin. The histogram corresponding to subimage SI is shown on the lower part of the drawing area.

function and activate a specific area with widgets and gadgets (i.e. push-buttons, text, togglebuttons, radioboxes). Colours and appropriate icons were chosen to minimize manipulation errors. The bottom side area is dedicated for image display control and for image zooming. Zooming allows an image to be enlarged by a factor of two. The operation concerns a selected quadrant of the image, of size 256 x 256 pixels (the display area is divided into four quadrants) to be enlarged to 512 x 512 pixels. The image display control uses push-buttons and scale widgets to modify the image appearance. Brightness, contrast, negative image and pseudocolor are capabilities. To display an image, 3

choices are offered to the user, we mostly use 80 grey levels, that gives a satisfactory image, 128 and 256 grey levels are the other choices. Functions Most of the IBIS function have already been presented with the original IBIS software (Flifla el ai, 1992). We present in this section the new features associated with IBIS v3. Loading Two kind of images can be loaded: raw images or IBIS

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Fig. 3. The quasi optical diffraction (QOD) procedure A selected image catalase is displayed in the top-left quadrant; its calculated power spectrum is automatically displayed in the adjacent quadrant Using the mouse, a numerical mask is carried out which keeps selected diffraction points (quadrant 3); the filtered image is then calculated by reverse Fourier transform and displayed (quadrant 4).

type images. To load a raw image its size must be specified, then maximum and minimum pixel intensities, average, standard deviation and histogram are computed. An IBIS type image has an image name followed with an extension *.ibsl or *.ibs8. These extensions are generated after any calculation procedure when a single image (*.ibisl) or a global eight sub-image (*.ibis8) is saved. During a processing sequence, when a new *.ibs8 image is loaded the last four sub-images displayed (nine to twelve) are not overwritten and thus can be used with the new images.

activated to execute this function. A windowing sequence is performed as follows: the user chooses a rectangular or a circular window, and selects an object in the main display area then drops it out in a selected position of the secondary drawing area. The maximum size allowed is respectively 120 x 120 pixels or a radius of 60 pixels. At each operation, the image average is calculated and a padding to 128 x 128 pixels is achieved. After the first windowing operation, IBIS allows cut off of the following sub-images using the same parameters.

Windowing

The quasi optical diffraction function

When a windowing function is called, a new display area is

Quasi optical diffraction (QOD), is a computer procedure

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FrankJ. Goldfarb.W., Eisenberg.D. and Baker,T.S. (1978) Reconstruction of glutamine synthetase using computer averaging. Ultramicroscopy, 3, 283-290. Franks., Shimkin.B. and Dowse,H. (1981) SPIDER a modular software system for electron image processing. Ultramicroscopy, 6, 343-358. Hegerl,R. and Altbauer,A. (1982) The 'EM' program system. Ultramicroscopy, 9, 109-116. Radermacher.M. (1988) Three-dimensional reconstruction of single particles from random and nonrandom tilt series. J. Electron Microsc, Tech., 9, 359-394. Radermacher,M., Wagenknecht.T.. Verschoor.A. and Frank.J. (1987) Three-dimensional reconstruction from a single-exposure, random conical tilt senes applied to the 50S ribosomal subunit of Escherichia coli. J. Microsc, 146, 113-136. Saxton,W.O.,Pitt,T.J. and Horner.M. (1979) Digital imaging processing: the SEMPER system. Ultramicroscopy, 4, 343-354. Smith,P.R. (1978) An integrated set of computer programs for processing electron micrographs of biological structures. Ultramicroscopv,3, 153160. Thomas,D., Flifla.M.J., Escoffier.B., Barray.M. and Delain.E. (1988) Image processing of electron micrographs of human alpha2-macroglobuhn half-molecules induced by Cd2 + . Biol. Cell, 64, 39-44. Trus.B.L. and Steven.A.C. (1981) Digital image processing of electron micrographs-the PIC system. Ultramicroscopy, 6, 383-386. Van Heel,H. and Keegstra,W. (1981) IMAGIC: a fast and friendly image analysis software system. Ultramicroscopy, 7, 113-130. Received on February 28. 1996, revised on April 11,1996. accepted on April 29. 1996

Discussion In this work we have introduced a new concept to IBIS image analysis software, a parameter checking before any calculation sequence, and a tool which displays appropriate messages in the multipurpose area. In order to keep track of the session, a copy of each successful operation is automatically saved in the IBIS system file, each short line including name of function and results are numbered. IBIS v.3 is modular and upgradable. The use of modular programming techniques helps considerably to add new calculation procedures and to modify sequences. Acknowledgements We wish to thank R.Razouki Al-Saigh for the user's guide, and F. Dagorn and J.De Poulpiquet, C.R.I., Universite de Rennes, for constructive help in OSF/Motif.

References Crowther,R.A., Henderson.R. and Smith.J.M. (1996) MRC Image Processing Programs. J. Struct. Bwl., 116, 9-16. Flifla.M., Garreau.M., Rolland.J.P., Coatrieux.J.L. and Thomas,D. (1992) IBIS Integrated Biological Imaging system • electron micrograph image-processing software running on Unix workstations. Comput. Applic. Biosci., 8, 583-586.

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equivalent to optical diffraction, an image analysis operation which is carried out on an optical bench. This method is used for image analysis to assess the quality of an image and its associated resolution, and for Fourier filtering, to separate the periodic information from the uniformly distributed noise. The procedure is illustrated in Figure 3. Quasi optical diffraction is performed on an image having a maximum size of 256 x 256 pixels: a given area is selected from a raw image, then padded to 256 x 256 if necessary. The result of each operation, i.e. windowing, power spectrum, selected mask, and filtered image is displayed into the four quadrants of the drawing area. From the power spectrum, an averaged radius profile line of the intensity is automatically calculated and displayed. The mask used for filtering is created interactively using the mouse button. Once the first point has been selected, and the hole size defined, its parameters are applied to the other selected points. Two kinds of masking can be performed: one uses the selected points, the other removes the selected points. The undo button allows cancelling of the last operation. The distance between two points in the power spectrum can be measured, and the result, in pixels, is displayed in the multipurpose area. At the end of a QOD sequence, the user can activate the undo button to start a new session with the same image. The user can save the result of one of these operations or all the operations. Magnifying is available in the QOD sequences.