The light microscope is widely used throughout the sciences to understand biological structure and dynamics. In particular optically sectioning light microscopy which allows internal three dimensional structure of living tissue to be visualized without physically pertrubing the organism has become the tool of choice for developmental biologists and embryologists.The noninvasive qualities of optical sectioning microscopy allow for the normal development of the organism while it is being imaged. This process of 4D imaging (3D imaging over time ) is widely available in several manifestions including Nomarksi imaging systems, confocal imaging systems, and multiphoton laser scanning systems. To meet the demands of our laboratory and the demands of our colleagues, we have developed a widely used 4D acquistion and viewing software program that allows scientists to acquire 4D data from opticaly sectioning microscopes. The applications for 4D microscopy continue to grow, fueled by several important developments, perhaps most significantly the completion of genomic sequences from several model organisms.

These remarkable endeavors are providing the complete parts lists of the target organisms. The primary sequence of every component protein of an organism can be deduced from these genomic sequences. This means that probes (such as antibodies) can be made to any protein and used to identify the location and distribution of that protein within an organism. The new challenge is to see how all these parts fit together and function as a living ensemble. Four Dimensional (4D) Microscopy, which uses a combination of optical sectioning microscopy together with computational techniques to study the structural dynamics of developing organisms, has become a powerful way to meet this challenge.

4D Microscopy is a important tool for understanding the process of embryogenesis. In C. elegans we have taken 4D sequences using Nomarski optics in order to determine cell lineages. The 4D microscopy suite was used to create the following movie of development of a wild type C. elegans embryo.

We can follow patterns of cell division using multiphoton microscopy to visualize Green Fluorescent Protein (GFP) labeled histones. The following movie is an animated stereo pair of a sequence of projections showing the dividing nuclei in a developing embryo. This sequence was generated using the 4D microscopy suite in conjunction with the wavelet based algorithms.

One significant application for 4D microscopy has been in understanding the process of cell fusion. The following movie shows the patterns of adherence junctions (visualized by GFP label) in a developing embryo. Cell fusions are identified by the disappearance of specific adherence junctions.

The sequence was generated by using the 4D microscopy suite in conjunction with the Vis5d software.