Creating The Embryo
The general functions of 3D-DIAS are 1) to obtain optical sections of a moving cell within a time period short enough so that the translocation of the cell between the first and last section is not significant, 2) to repeat the reconstruction process at short enough time intervals so that the behavioral changes of interest can be analyzed, 3) to reconstruct not only the 3D surface of the cell, but also subcellular compartments, zones, vesicles, vacuoles and molecular complexes, 4) to view the reconstructions dynamically (as a time series movie) in 3D on a stereo workstation, and 5) to compute 3D motility and dynamic morphology parameters of the whole cell as well as each subcellular compartment.
Acquiring Optical Sections for 4D Reconstructions
Optical sections are generally obtained at a rate of 30 per second (video rate) and this process can be repeated at intervals as short as every second. A microstepper motor moves the focal plane through a desired distance either at a constant rate or through steps. Optical sections are digitally captured into 3D-DIAS. A character generator marks each optical section for the direction of scan.
Automatic outlining is based on a measurement of complexity for each pixel-neighborhood on the screen. An algorithm of distinguishing continuous edge pixels from interior singular pixels having high standard deviations allows discrimination of the cell perimeter from interior detail.
In this figure, twelve of a set of 30 optical sections of a Dictyostelium amoeba obtained within a two second period, at one micron intervals, and the auto-traced perimeters of the in-focus portions of the cell images are presented. Then the ‘caged’ reconstructions are shown at various angles of view.
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