Imaging Through the Pupal Case of <em>Drosophila melanogaster</em>

Abstract

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues. Video LinkThe video component of this article can be found at

Multi-view confocal microscopy enables multiple organ and whole organism live-imaging

“…Spinning disk confocal microscopy has often been employed to image individual epithelial tissues in Drosophila such as for example the wing, notum and the abdomen (Aigouy et al, 2010;Arata et al, 2017;Curran et al, 2017;Diaz-de-la-Loza et al, 2018;Etournay et al, 2015;Founounou et al, 2013;Gho et al, 1999;Guirao et al, 2015;Kanca et al, 2014;Keroles et al, 2014;Levayer et al, 2015;Michel and Dahmann, 2020;Prat-Rojo et al, 2020;Ray et al, 2015). Since individual tissues could successfully be imaged using confocal microscopy, we hypothesized that sample rotation along the anterior-posterior axis in combination with confocal microscopy could enable the visualization of multiple tissues.…”