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or
4
animals without the complication of autofluorescence. Imaging in lambda mode
should therefore yield signals that conform solely to the GFP emission spectrum.
3.3
VISUALIZATION OF LIPID DROPLET ASSOCIATED
PROTEINS BY SPINNING DISK CONFOCAL MICROSCOPY
3.3.1
Background
A spinning disk confocal microscope, when coupled with a sensitive charge coupled
device (CCD) camera, can exceed the sensitivity of a laser scanning confocal micro-
scope system. The fast acquisition time of a spinning disk confocal microscope also
allows the collection of three- or four-dimensional data that may not be possible on
other systems. For example, it takes seconds rather than minutes to acquire data of a
15-
m field of
view). The actual acquisition time is dictated by the intensity of the fluorescence sig-
nal, which depends on protein concentration and laser power, and the sensitivity of
the camera. Since the immobilization of live animals may not be complete, fast
acquisition time also minimizes the probability of sample movement during image
acquisition. This in turn allows high quality reconstruction of all signals in a three-
dimensional space. The use of spinning disk confocal microscopy is instrumental for
comprehensive quantitative measurement of lipid droplet size in a given cell, using
GFP::DGAT-2 or mRuby::DGAT-2 as markers. The diameter of individual lipid
droplets can be accurately measured in a three-dimensional rendering program such
as Imaris (Bitplane).
m
m
Z
-stack with individual slices separated by 0.25
m
m (68
m
mby68
m
3.3.2
Methods
Animals are mounted onto glass slides following the same procedures described in the
previous section. L4 larval animals are imaged on a PerkinElmer Ultraview spinning
disk confocal microscope, equipped with a 100
, NA1.45 oil Plan-Apochromat ob-
jective and a Hamamatsu Orca-R2 CCD camera. The system is controlled by the Volo-
city software (PerkinElmer). For GFP, a 488-nm laser is used for excitation and signals
are collected with a 500-555-nm emission filter (
Fig. 3.3
A). For mRuby, a 561-nm
laser is used for excitation and signals are collected with a (415-475 nm)
(580-650 nm) dual-pass emission filter. To capture autofluorescence from LROs, a
488-nm laser is used for excitation and signals are collected with a (415-475 nm)
(580-650 nm) dual-pass emission filter (
Fig. 3.3
B). The output power of each laser
is adjusted to avoid bleaching. Optical sections were taken at 0.25-
m
m intervals.
3.3.3
Discussion
Linear unmixing cannot be easily implemented in a conventional spinning disk con-
focal system. One challenge is to distinguish GFP versus autofluorescent signals that
are collected through the 500-555-nm emission filter (
Fig. 3.3
A). Our strategy is to