Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 388

Fig. 4 Imaging of F-actin during morphogenesis. (A, B) F-actin dynamics during ventral enclosure
imaged in a wild-type embryo expressing vab-10::ABD::GFP. Images were acquired using a Perkin-
Elmer UltraView LCI system, with Hamamatsu Orca II-ER camera. The boxed regions in (B) show
extensive actin accumulation between two pairs of ventral epidermal cells. Elapsed time between (A) and
(B) is 1380s. (From Lockwood et al., 2008 ). (C) Phalloidin staining of an elongated embryo.
Circumferential filament bundles (CFBs) are clearly visible throughout the epidermis. The arrow points
to junctional actin. (From Costa et al., 1998 ). (D) A twofold stage embryo expressing VAB-10ABD::GFP.
CFBs are prominent; arrow points to junctional actin. Image courtesy of R. Zaidel-Bar. Bars = 5 m m.
several key factors affecting 4D fluorescence acquisition during morphogenesis.
First, as with 4D Nomarski imaging, ambient temperature must be controlled care-
fully. The temperature must be kept below 25 C; for long films, a temperature closer
to 20 C is advisable. This is often not possible in shared user facilities, in which
elevated temperatures suited to tissue culture work are the focus. Second, despite
theoretical calculations of voxel sampling in Z stacks of fluorescent images, it is
typically advisable to acquire very closely spaced optical sections if one is imaging
events in the epidermis. We have found that focal planes spaced 0.5 m m apart or less
are necessary, due to the extreme thinness of the epidermis. Finally, modern lenses
with newer coatings make a significant difference. For very high-resolution filming
of cytoskeletal elements or thin structures, we have found that very high NA lenses
are helpful. In particular, we have found that lenses designed for total internal
reflection microscopy (TIRF), but without the internal optics for TIRF itself, provide