Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 397

3. Importing data from the confocal microscope

a. Invoke the LOCI Bio-Formats Importer plugin within ImageJ: Many standard

installations of ImageJ, or the package installer based variant of ImageJ, Fiji
( http://pacific.mpi-cbg.de/wiki/index.php/Fiji ) may have Bio-Formats
Importer installed, or it can be obtained from http://www.loci.wisc.edu/soft-
ware/bio-formats . The Bio-Formats plugin can read many data formats,
including most raw image formats generated by standard confocal micro-
scopes. We have tested this code using datasets from an Olympus Fluoview
1000 (.oif format files), but the basic approach should work for other types of
datasets.

b. Import the dataset as an image stack into ImageJ: After opening the Bio-

Formats plugin, select a file within the directory containing the dataset, and
import the data. Our setup uses a SIM scanner for photobleaching. The frame
in which the bleach is applied often has high background. This frame can be
deleted. The Bio-Formats plugin also often imports a few extraneous files into
the stack that should be deleted before proceeding.

4. Selecting ROIs for FRAP analysis

a. Open the ROI Manager in ImageJ: Once the stack has been opened and
cleaned up, open the ROI Manager [ImageJ ! Analyze ! Tools ! ROI

Manager].
b. Define bleach zone ROI: Navigate to the frame immediately after the bleach
pulse was applied. Select the elliptical marquee tool in ImageJ from the main
ImageJ window, and drag out a ROI that encompasses the bleach region. Once
selected, add it to the ROI Manager list by typing [t].
c. Define control ROI: A second ROI must be selected which serves as a control
for the overall bleaching of the specimen during postbleach filming. This ROI
can be a large region of the embryo. For FRAP of junctions, we typically select
a large, but comparable, region containing the unbleached junctions. Once
selected, add it to the ROI Manager list by typing [t].
d. Select the bleach zone ROI in the ROI Manager list to reselect it.

5. Output of the plugin

1. Run the FRAP Profiler plugin [ImageJ ! Plugins ! (location where the plugin
was installed)]. A dialog will appear with pull-down menus for selecting
various options. These include:

i. Single or double exponential curve fitting. Choose the latter if it appears,
or there is physical reason to suspect, a two-step recovery process.
ii. The real-world time interval between successive images in the movie.
iii. The option to produce an image showing the bleach ROI and the control
ROI overlaid on the first postbleach image.