Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 395

3. FRAP
Introduction
In addition to imaging specific structures within embryos during morphogenesis,
fluorescence can also be used to study the dynamics of redistribution of molecules in
embryos using FRAP. GFP can be used very successfully in studies involving FRAP
( Lippincott-Schwartz et al., 2003 ), which is easily done via repeated scanning of a
selected ROI in the LSCM. FRAP has been used in several contexts in C. elegans,
particularly in one-celled zygotes (e.g., Labbe et al., 2004 ).
Methods
1. Acquiring FRAP data
The acquisition of the raw data to be analyzed in a FRAP is highly dependent on
the apparatus and acquisition software, so it will not be discussed here in detail.
We have been able to successfully photobleach single junctional domains in
embryos undergoing morphogenesis. In our case, these experiments have been
performed using an Olympus Fluoview 1000 with SIM scanner for rapid photo-
bleaching with simultaneous imaging ( Fig. 7A,B ). In general, the conditions that
allow for successful imaging via laser scanning confocal microscopy pertain. We
have found that single focal planes can be imaged in embryos in agar mounts
quite successfully. We have also found several specific considerations to be
important in performing FRAP during morphogenesis:
a. During morphogenesis, embryos may move/twitch slightly. The bleach zone
ROI must be chosen such that the bleach zone does not move out of the ROI.
For FRAP of junctional GFPs, this can be accomplished by altering the size
and/or aspect ratio of the ROI. Note that as a result, FRAP is practically
limited to early phases of elongation; after this time, embryos twitch too much
to make reliable measurements.
b. Embryos in which FRAP is performed at temperatures exceeding 27 Cmay
show erratic results. This is likely due to overall sickness of embryos at
elevated temperatures. If possible, filming should occur at 25 C.
2. Analyzing FRAP data: Simplified FRAP analysis using ImageJ
Once obtained, analysis of the recovery kinetics of a bleached region in a FRAP
experiment proceeds along similar lines. There are many options for analyzing
data from FRAP experiments. The technique we describe here is a free, semi-
automated solution suitable for use by undergraduates, and relies on plugins we
have adapted from original Java routines written by Tony Collins, McMaster
University ( Collins, 2007 ). This plugin is available for download from http://
worms.zoology.wisc.edu/research/4d/4d.html . To use, install the plugin by copy-
ing to a convenient directory within the Plugins directory in ImageJ. For more
information, see the ImageJ web site ( http://rsbweb.nih.gov/ij/ ).