Stereo-4-D reconstruction and animation from living fluorescent specimens

We present a novel approach to the viewing and analysis of 4-dimensional (4-D) data sets recorded from live fluorescent samples. With stereo-4-D reconstructions, the observer manipulates a rotatable projection of the full 3-dimensional (3-D) specimen while simultaneously controlling animation of the recording forward or backward in time. The result is a unique lifelike perspective on the development of an entire living subject. Here, we apply this technique to the observation of the cell membranes of developing Caenorhabditis elegans. Embryos labeled with the vital plasma membrane probe FM 4-64 were imaged by multiphoton laser scanning fluorescence microscopy, yielding 4-D data sets of entire embryos over several hours of development. Stereo 4-D and standard focal-plane 4-D viewing of these novel time-lapse recordings provide the observer with detail at both the subcellular and whole-animal level from a single data set and produce a unique record of the lineage, cell shape changes, cell contacts and morphogenetic dynamics that make up embryogenesis. The procedures by which stereo-4-D reconstructions are created and viewed rely on public domain software running on a personal computer and should therefore be accessible by a general audience. Data output utilizes the versatile and well-supported QuickTime animation format. Additional features allow for stereo-4-D reconstruction of isolated 3-D volumes of interest from within the larger specimen.