Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 385

vii. These mounts can be imaged using 4D microscopy. We have also found it
possible to remove the coverslip and process embryos for phalloidin stain-
ing (see above; for examples, see references).
viii. Embryos can be scored after 4 h for blue gut granules, which indicate that
sufficient permeabilization was achieved for Nile Blue A penetration.
Discussion
Performing long-term 4D filming after perforation of the eggshell is difficult.
Many embryos show abnormalities in subsequent development. Extensive negative
controls (i.e., perforation of the eggshell in the presence of carrier, such as DMSO,
alone) are therefore highly advisable. If sufficient precautions are taken, however, it
is possible to perform pharmacological inhibition followed by 4D filming, as we
have shown in several circumstances ( Thomas-Virnig et al., 2004; Williams-Masson
et al., 1997 ).
C. Fluorescence Imaging of Morphogenesis
1. Introduction: Imaging Modalities for 4D Fluorescence Imaging
Nomarski microscopy, while a daily workhorse for imaging morphogenesis and
performing basic phenotyping, is limited. Refractile elements in the cytoplasm of
embryonic cells, combined with the inherent curvature of the embryo, limits the
resolution of the standard Nomarski microscope. In addition, the epidermis is
exceedingly thin (less than 0.5 m m in some cases), making it difficult to resolve.
Fluorescence imaging of specific structures in embryos, combined with confocal or
multiphoton microscopy, overcomes these challenges. The chapter by Maddox and
Maddox in this volume covers basic modalities of fluorescence microscopy. Here we
discuss several useful strategies for visualizing cells and subcellular structures
during morphogenesis, an alternative phalloidin staining procedure, and a simple
strategy for analyzing fluorescence recovery after photobleaching (FRAP) data.
Given the wide array of genetically encoded fluorescent probes avaialble (see
below), 4D datasets of fluorescent specimens acquired using confocal, multiphoton,
or widefield deconvolution techniques have several advantages over 4D datasets
acquired using transmitted light optics, such as Nomarski microscopy. First, such
techniques permit much more refined optical sectioning of the specimen with little
contribution by out-of-focus information. Secondly, it is much easier to understand
the distribution of the fluorescent signal from a 3D reconstruction of a sample than a
3D-stack of DIC images (for an attempt at the latter, see Heid et al., 2002 ). For thin
specimens imaged within 5 m m of the coverslip such as C. elegans embryos, oil
immersion optics and a high NA lens (NA = 1.4-1.45) are typically the best choice.
Although the use of fluorescent probes present several key advantages, it also
presents several challenges for 4D imaging of morphogenesis. Because C. elegans