In order to obtain measurements of the surface areas, lengths and volumes of the three-dimensional biological structures contained with volume data derived by electron tomography, it is necessary to define the surface of the structure. Based on an extensive analysis of volume based image processing methods such as those availbale in the program, ANALYLZE, we found that surfaces could not be segmented automatically due to inhomogeneities in the staining of the structures and limitations in the resolution of tomographic reconstruction of large extended structures. In agreement with studies by other researchers, we have found that the most accurate definition of the location of surface membranes is obtained using an interactive manual tracing method. We developed a program, xvoxtrace, for manually contouring structures in 2-D orthogonal slices of a 3-D tomographic dataset. Using this program, the researcher draws surface outlines, using a mouse or digitizing tablet, which appear on a display of the slice. As an essential guide to defming the surface, the user simultaneously observes the tracing on selected images from the tilt series used to derive the volume and also on a volume rendering of the structure. To localize the contours in three-dimensions, the tilt series and volume views may be observed in stereo. By drawing contours in successive slices from the volume, a 3-D cross-sectional representation of the structure is defined. During this past year, xvoxtrace has been used extensively in the Resource and in the laboratories of collaborators to whom this program has been distributed. On the basis of reccomendations from these users, a new improved version of the program is under development and will soon be completed. This version will include enhanced contour editing facilities, such as automatic contour resplining, and capabilities for panning and zooming in on a view of a selected region of the specimen in the volume slice, the volume rendering, and the tilt display. We have also developed xdend an interactive analysis tool for measurement of volumes, surface areas, and lengths of contoured objects, primarily designed to postprocess xvoxtrace data. Objects may be resectioned along the transverse, saggital, or coronal axes to optimize the accuracy of measurement. In addtion, objects may be viewed in perspective as contour outlines or as centroids or skeletons.
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