Biology Reference
In-Depth Information
viii. Cut 0.5-
m
m sections and place on 3-APTS coated coverslips. To coat 12-mm
round coverslip with 3-APTS: clean 12-mm round coverslips in detergent
solution. Rinse 10
in dH2O. Dehydrate using three changes in absolute
ethanol. Coat slides in 2% solution of 3-APTS in dry acetone for several
minutes. Rinse twice in dH2O. Spread out coverslips on clean filter paper and
dry at room temperature. Stain thick sections with Richardson
'
sstain.Rinse
coverslips gently after staining with distilled water. Cut thin and/or semithin
sections after embryos are detected in thick sections by light microscopy.
d. TEM
Collect thin sections on formvar-coated slot grids to provide an unobstructed
view of an entire section. Stain thin and ultrathin sections for 10-20 min in 1%
aqueous uranyl acetate, followed by 3 min in Reynolds
'
lead before viewing in a
TEM. Thinner sections may require longer staining times. Large montage images
can be collected manually and ''stitched together'' in Photoshop (Adobe) or
collected and montaged automatically with analySISTM or similar software.
When comparing different embryos within the same mount, it is helpful to make
a map using a transmitted light image of the embedded group of embryos.
Number the ''mutant'' and rescued embryos to keep track of higher magnifica-
tion images. Because a TEM image may be a mirror image of the transmitted
light image, flip the image in Photoshop with the same numbering scheme to be
prepared for either orientation.
e. Overlaying Correlative Microscopic Images
A single transmitted light image is typically chosen from the multiple focal
planes acquired based on features of interest that were in focus. The fluorescent
image is a brightest point z projection (ImageJ) of all fluorescent images
acquired. Because embryos are alive at the time of image capture, and hence
may be moving, the fluorescent image(s) may be blurred. The detection of GFP
confirms the presence of the rescuing DNA and definitively identifies the geno-
type of embryos. Confocal fluorescent and transmitted light images are manip-
ulated with ImageJ and Adobe Photoshop. LSCM images are prepared using
ImageJ. Some of the light (LSCM) and EM images collected may be mirror
images of each other; they can be corrected in Photoshop. LSCM and TEM
images are aligned starting with the TEM image, which usually contains more
pixels.
Fig. 8
demonstrates the results of this correlative method.
III. Summary
C. elegans embryos are a powerful model system for imaging detailed cell move-
ments, intercellular dynamics, and the overall shape changes that occur during
morphogenesis. With the widespread use of genetically encoded fluorescent markers
and the ability to perform correlative electron microscopy on transgenic embryos,
the virtues that originally led to the selection of C. elegans as a model organism have
been expanded. In the future, other extensions of live imaging, such as
in vivo
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