Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture

One of the overarching goals in developmental biology is the elucidation of mechanisms that elaborate form and function. To this end, an accurate morphological description of embryonic development is essential. However, visualizing dynamic changes in the three-dimensional (3D) structure of the developing embryo has been a "holy grail" in the field of developmental biology. The fundamental difficulties that have hindered all efforts in 3D reconstruction using two-dimensional (2D) image stacks revolve around the seemingly intractable problems of section registration and distortion. A remarkably simple solution has come about with the development of a new technique referred to as episcopic fluorescence image capture (EFIC). With EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting each section. The 2D resolution obtained is close to that achieved by histology, and such 2D image stacks can be readily reconstructed in 3D. The 3D models generated provide fine structural details with resolution unmatched by 3D reconstructions obtained with any other imaging modalities. Given the perfect registration of EFIC image stacks, another important capability provided by EFIC is digital resectioning in any plane. This provides complete flexibility in the selection of optimal virtual sectioning planes for viewing different features in a specimen, and is invaluable for analyzing dynamic changes in tissue structure in the developing embryo. The capabilities provided by EFIC for rapid high resolution 3D reconstruction together with digital resectioning make this an unparalleled tool for characterizing morphogenetic events in the developing embryo. Although our review is focused on using EFIC for studying embryonic development, it is important to note that there is no intrinsic limitation on the size of the specimen that can be analyzed by EFIC imaging. Overall, EFIC should serve as an important imaging technique that will complement other 3D imaging modalities such as MRI and optical tomography. Given the feasibility of generating EFIC image stacks using cryoembedded or polyethylene glycol (PEG)-embedded specimens, there is the possibility that EFIC may be combined with 3D RNA or protein expression profiling. Together, such studies may help further elucidate the relationship between form and function.