Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 386

embryos are not flat, multiple focal planes must be acquired at each successive
time point, requiring repeated exposure of the region of interest (ROI) to excita-
tion photons that compromise embryo viability. In addition, the photobleaching
that accompanies repeated observation in 4D experiments complicates image
quantification. If photobleaching is not too severe, it is sometimes possible to
normalize the signal from bleached specimens using several algorithms (e.g., see
the ImageJ plugin that is part of the McMaster Biophotonics suite of routines:
http://www.macbiophotonics.ca/imagej/ ).
Several imaging modalities have been used frequently for analyzing morphogen-
esis. For the physical basis of each, readers should consult the chapter by Maddox
and Maddox in this volume. Here we will focus on issues related to filming
morphogenesis.
Laser scanning confocal microscopy (LSCM)
In some cases LSCM suffices. Depending on the density of fluorophore labeling
and the nature of the tagged moiety, many experiments have been performed using
rather unremarkable equipment, such as a Bio-Rad 1024 or similar older laser
scanning confocal devices. Interested readers are urged to consult older reviews
describing methods of confocal imaging in C. elegans (e.g., Mohler, 1999 ). LSCM is
typically the mode of choice for performing single focal plane photobleaching
experiments (see below). For long-term 4D acquisition in which many focal planes
are acquired, LSCM quickly leads to arrest of embryos. The time required to induce
arrest varies, depending on the ambient temperature and the probe being imaged.
However, in general LSCM is not well suited to long-term viability of embryos
filmed over many hours.
Multiphoton excitation laser scanning microscopy (MPLSM)
Multiphoton laser-scanning microscopy has several potential advantages over
LSCM for live imaging of embryos. MPLSM excites fluorescence using a series
of short, high-energy pulses of near-infrared photons from a mode-locked laser.
MPLSM has a key advantage for live embryo imaging experiments in C. elegans. In
a two-photon microscope the probability of excitation varies as the inverse fourth
power of the distance from the focal plane. Photons are thus only absorbed in a very
small volume centered on the plane of focus, eliminating photobleaching and photo-
damage caused by excitation of fluorophores above and below the plane of focus.
The resulting improvements in viability can be quite dramatic. In our laboratory, C.
elegans embryos expressing a GFP-tagged junctional protein survive for 30-90 min
when imaged using a Bio-Rad 1024 CLSM at low power (10%; J. Hardin, unpub-
lished) but the same embryos can be imaged for many hours using MPLSM
( Raich et al., 1999 ). An example is shown in Fig. 3 (see Koppen et al., 2001 for
more details).