3-D Movie Rendering of NFR-labeled Microplasmodium

Volume rendering of a Physarum polycephalum microplasmodium (MP) in which the nuclei were labelled with Nuclear Fast Red (NFR). The MP was imaged by confocal microscopy and a Z-series data set was used to construct the movie shown here. The two still frames shown above (and taken from the movie sequence) show the MP (which is adherent to a coated glass slide) seen from above (left) and from below (right - i.e. from the side that is in contact with the glass slide). The movie shows the MP as it would appear if rotating about an axis running parallel to the surface of the slide and the y-axis of the screen.

Details: Physarum microplasmodia growing in standard suspension culture were fixed, applied to specially-coated glass slides, permeabilized, and stained with NFR according to procedures presented, in preliminary form, at the Physarum Meeting 2002 in Freiburg im Breisgau, Germany (July 2-5, 2002). The NFR staining, which is still being characterized, labels nuclei and, to a variable extent, the extracellular slime coat of the MP. The data shown here was recorded, during the experiment of 5/29/02 (extended on 6/14/02), as "Zser4.word", using a BioRad MRC600 confocal microscope system interfaced with a Zeiss Axiovert light microscope, employing a 63X oil immersion lens. The Z-series (90 optical sections at 0.46 um intervals, with the confocal aperature set to minimum) was recorded at a zoom factor of 1.3; the full frame width of the images used for this rendering was 140 um. It should be noted that the process used to "stick" the fixed MP to the slides causes two artefacts: first, the MP tend to flatten to a variable degree (MP of the size shown here are usually spherical when observed in suspension) and second, the adhesion to the substratum is such that staining of the external slime coat on that side is greatly reduced (compared to the "upper" portions of the MP). Using BICViewer* software, the Z-series was processed to create a 39-frame movie: a series of "maximum intensity" projections, the sum of which shows a total rotation, about the y-axis, of 360 degrees. This 39-frame stack was then converted to a set of 39 individual *.tif files, which were ported to a Macintosh G-3 running under OS X. Tiffany software was used to enhance and pseudocolor the *.tif files and convert them to *.gif files. These *.gif files were then converted to an animation using GifFun software. It should be noted that BICViewer* software was used to apply an arbitrary degree of "fading" from the "top section" to the "bottom section" of the Z-series, which enhances the 3-D appearance of the rendering shown here; in reality, the nuclei in all sections of the actual Z-series show essentialy the same degree of fluorescence.

* I am especially grateful to Dr. Jozsef Czege for writing the BICViewer software. In response to my request for software that would run "like Confocal Assistant, only under OS-X", he created a far more powerful and sophisticated image processing and volume rendering software package than I would have ever imagined.

Last updated November 1, 2002.