Multi-View Imaging of Drosophila Embryos

Abstract

There are several reasons for imaging a single, developing embryo from multiple view points. The embryo is a complex biomechanical system and morphogenesis movements in one region typically produce motions in adjacent areas. Multi-view imaging can be used to observe morphogenesis and gain a better understanding of normal and abnormal embryo development. The system would allow the embryo to be rotated to a specific vantage point so that a particular morphogenetic process may be observed clearly. Moreover, a multi-view system can be used to gather images to create an accurate three-dimensional reconstruction of the embryo for computer simulations. The scope of this thesis was to construct an apparatus that could capture multi-view images for these applications. A multi-view system for imaging live Drosophila melanogaster embryos, the first of its kind, is presented. Embryos for imaging are collected from genetically modified Drosophila stocks that contain a green fluorescing protein (GFP), which highlights only specific cell components. The embryos are mounted on a wire that is rotated under computer control to desired viewpoints in front of the objective of a custom-built confocal microscope. The optical components for the orizontallyaligned microscope were researched, selected and installed specifically for this multi-viewing apparatus. The multiple images of the stacks from each viewpoint are deconvolved and collaged so as to show all of the cells visible from that view. The process of rotating and capturing images can be repeated for many angles over the course of one hour. Experiments were conducted to verify the repeatability of the rotation mechanism and to determine the number of image slices required to produce a satisfactory image collage from each viewpoint. Additional testing was conducted to establish that the system could capture a complete 360° view of the embryo, and a time-lapse study was done to verify that a developing embryo could be imaged repeatedly from two separate angles during ventral furrow formation. An analysis of the effects of the imaging system on embryos in terms of photo-bleaching and viability is presented.