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4.2.2 Imaging LDs in fixed fat bodies using confocal microscopy
The effects on LD morphology from knocking down any fly genes can be assessed
with confocal microscopy ( Fig. 4.2 B). After wandering third instar larvae are col-
lected, their fat bodies are dissected and briefly fixed in 4% paraformaldehyde/
1 PBS for 10 min. LDs in the fat body are stained with 2 m g/ml BODIPY493/
503 for 5 min. Prolonged fixation (i.e.,
24 h) might alter
LD morphology in the fixed fat bodies. Thus prompt examination under a confocal
microscope is essential to accurately recapitulate the status of LDs.
30 min) or staining (
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4.2.3 Imaging LD dynamics in vivo using coherent anti-stokes
Raman scattering microscopy
Coherent anti-Stokes Raman scattering (CARS) microscopy has developed in recent
years as a very promising imaging platform for long-term, noninvasive and label-free
observation of LD dynamics in cells and developing animals ( Min, Freudiger, Lu, &
Xie, 2011 ). CARS microscopy employs the property of the Raman scattered photons
exhibiting molecular vibration specific frequency shifts. It is very well suited for
detecting lipid-rich cellular structures (such as LDs) using a spectral beating fre-
quency matching that of the symmetric stretch vibration of CH 2 groups abundant
in fatty acid side chains.
Although CARS microscopy can be used to image fixed samples, its greatest
advantage is obvious when used for live samples without any dye labeling (which
might cause LD morphology changes by itself or the solvent in which it is dissolved,
Fukumoto & Fujimoto, 2002 ). Because the emission spectrum of CARS falls within
a wide range overlapping with that of the red and far-red fluorescent proteins, we
avoid using those fluorescent proteins in the samples for CARS imaging. We used
an Olympus two-photon microscope equipped with an XLPlanN 25x/1.05w MP
objective lens, and the Mai Tai DeepSea laser (Spectra-physics) was tuned to 800 nm.
Specifically, we dissect larval fat bodies from wandering third instar larvae and
keep the freshly isolated fat bodies in PBS sandwiched between a microscope slide
and a coverslip. This mini imaging chamber is made by using two coverslips on the
sides as spacers and sealed by nail polish on all four sides. One should avoid applying
physical force on the samples during mounting as it will cause LD fusion and change
the morphology. Dissected fat bodies can survive in this imaging chamber for at least
a few hours, allowing detailed morphological studies of LDs with CARS microscopy
in a label-free manner ( Fig. 4.2 C).
CARS microscopy has been used recently to image LDs in anesthetized, intact
second instar larvae ( Chien et al., 2011 ). The size and physical viscidity of third in-
star larvae makes it harder to image the fat body within the live larvae. The pupal
stage of Drosophila development is immotile. For this reason, it offers the optimal
time window to observe long-term changes of LD dynamics within intact live ani-
mals. Because the pupal case interferes with imaging due to its lack of optical trans-
parency, we hand peel off the pupal case with a pair of fine tweezers to prepare
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