Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 387

Fig. 3 Comparison of Nomarski 4D imaging (A, C, E, G) with the use of DLG-1::GFP as a junctional
marker (B, D, F, H). (A, B) Early dorsal intercalation of epidermal cells. (C, D) Intercalation complete.
(E, F) Early ventral enclosure. (G, H) Ventral enclosure complete. Bar = 5 m m.
(Adapted from Chisholm and Hardin, 2005 ).
Although MPLSM can be superior to CLSM for many applications, there are
several issues that can make MPLSM less than optimal. First, the typical MPLSM
device is expensive, placing it out of reach of most individual laboratories. In
contrast, individual labs can often afford disk-scanning confocal microscopes, a
factor that is particularly important for live embryo studies, which often monopolize
microscope time. Second, for fluorophores that emit in the red portion of the visible
spectrum, the wavelengths needed to generate a two-photon event are longer than
those produced by the Ti:Sapphire lasers commonly used in commercial MPLSM
devices. For such probes, Nd::YLF lasers, which emit at 1047 nm, are very effective
( Mohler and White, 1998 ), but may not be readily available.
Disk-scanning confocal microscopy
For the developmental biologist, spinning disk systems based on Yokagawa scan-
heads are an inexpensive alternative that provides many of the benefits of more
elaborate technologies, such as MPLSM. Because disk-scanning systems use an off-
the-shelf focus motor, CCD, filter wheel, and shutter components, commercial
imaging packages or freeware packages such as Micro-Manager can be used to drive
data acquisition. In our laboratory, disk-scanning technology has largely replaced
both CLSM and multiphoton microscopy for routine 4D data acquisition during
morphogenesis (for examples, see Figs. 4A,B, 5, and 6B,C ). The data generated by
spinning disk can also be improved via postacquisition deconvolution, making its Z
resolution comparable to LSCM or MPLSM.
Considerations for optimizing 4D data during morphogenesis
There are several thorough treatments of empirical considerations that lead to
high-quality 4D fluorescence dataset (e.g., Hardin, 2006 ). Here, we will focus on