3-D Movie Rendering of Actin-labeled RAFT Cell

Volume rendering of Stage-III Amoeboflagellate of Physarum polycephalum in which polymeric (F-) actin was labelled with Rhodamine-Phalloidin. The cell was imaged by confocal microscopy (BioRad MRC600) and the Z-series data set was used to construct the movie shown here. The cell is seen with its long axis parallel to the substratum and oriented approximately in the "y" direction, with the anterior of the cell (A - anterior flagellae not seen) at the top of the field. The dark oval space (*) reflects the absence of actin from the nucleus. Actin is concentrated in a flattened projection (the "ridge" = R) extending from one side ("dorsal") of the cell, as well as in a cluster of "posterior spots" (P) that correspond to the point at which the transforming cell makes transient adhesions with the substratum. The movie clip begins with the cell viewed as in the upper left figure, shows the cell "rotating" to the orientation in the upper right figure, and then - after a slight pause - rotating back to the beginning of the animation sequence.

Details: Physarum amoebae undergoing the Repeat AmoeboFlagellate Transformation (RAFT) were fixed, stained with Rhodamine-Phalloidin, and processed for light microscopy according to Pagh and Adelman, 1988. The data shown here was recorded, during the experiment of 6/1/93, as "boxzser2.pic", using a BioRad MRC600 confocal microscope system interfaced with a Zeiss Axiovert light microscope, employing a 63X oil immersion lens. The Z-series (18 optical sections at 0.5 um intervals) was recorded at a zoom factor of 4; the full frame width of the "boxed" file used here was 19 um. Using Confocal Assistant software, the Z-series was processed to create a 60-frame movie: a series of "maximum intensity" projections, the sum of which shows a total rotation, about the y-axis, of 180 degrees (hence each frame is "rotated" 3 degrees relative to the preceding one). This 60-frame stack was then converted to a set of 60 individual *.tif files, which were ported to a Macintosh G-3 running under OS X. Tiffany software was used to pseudocolor the *.tif files and convert them to *.gif files. [In addition, "labeled" versions of the first and last files were made, for use as the "stills".] These *.gif files were then converted to animations using GifFun software For the "detailed" version shown here, the 60 gif files were duplicated and the duplicate set was renumbered to create a "return to original orientation" segment so that the animation contains a total of 120 frames and depicts the cell being rotated from 0 to 180 degrees and then rotated smoothly back.

Last updated March 26, 2002.