Biology Reference
In-Depth Information
background fluorescent signals in the media so that only signals from the fluoro-
phores incorporated into LDs are collected. For this purposes, labeling medium B
should be added into the culture chamber on the microscope, immediately before
image capture (see
Section 2.3.3
).
In principle, the choice of fatty acid analogues can be reversed, that is,
BODIPY FL C
12
for preformed and BODIPY 558/568 C
12
for nascent CLDs. We
have demonstrated by both microscopy and TLC that these analogues are
similar in their capabilities to incorporate into TG/LDs. However, we prefer using
BODIPY FL C
12
for nascent LD formation in live-cell imaging for the following
reasons: (1) compared to BODIPY 558/568 C
12
, BODIPY FL C
12
gives brighter
signal and sharper image; (2) as a consequence, lower laser power can be used
for live-cell imaging, which is crucial to keep cells healthy during imaging and
for avoiding artifacts in quantification due to bleaching of the fluorophore; (3)
chemical quencher for BODIPY FL C
12
is readily available in the commercial kit
mentioned above.
7.2.3.3
Microscope setup and imaging
The spinning-disk confocal microscopes suitable for this experiment should be
equipped with green and red laser lines for excitation of the fatty acid analogues.
It is essential to have a temperature-controlled environment chamber. It is preferable
to have CO
2
supply; however, cells can be maintained in medium containing 50 mM
HEPES for at least 30 min without CO
2
. Samples should be secured to the stage with
an adaptor that completely restricts movement of the samples when reagents are
added during imaging. For this reason, a culture chamber is preferable to a glass bot-
tom dish, as the latter usually does not fit tightly to the stage adaptor. It is also ex-
tremely helpful to have multistage control and autofocusing to image multiple cells
during the same imaging session. Taking Z stacks of cells is helpful to bypass focus
fluctuations during long-term imaging.
For our setup, cells grown on cover slips are mounted onto a culture chamber
(Chamlide, Seoul, Korea) and placed in an environment chamber thermostated at
37
C and supplied with 5% CO
2
. Labeling media B is carefully added to cells in
the culture chamber immediately before image capture (time 0). Confocal micros-
copy is performed on a spinning-disk microscope (WaveFx from Quorum Technol-
ogies, Guelph, Canada) setup on an Olympus IX-81 inverted stand (Olympus,
Markham, Canada). Images are acquired through a 60
objective (N.A. 1.42) with
an EMCCD camera (Hamamatsu, Japan). Fluorescent fatty acid analogues BODIPY
FL C
12
and BODIPY 558/568 C
12
are successively excited by a 491 nm (GFP chan-
nel) and a 543 nm (Cy3 channel) laser line (Spectral Applied Research, Richmond
Hill, Canada), respectively. Z-slices of 0.5
m steps are acquired using Volocity soft-
ware (Improvision) through the cells using a piezo z-stage (Applied Scientific Instru-
mentation, Eugene, USA) with image capture every 1 min over a period of 30 min.
Quantification of fluorescent intensity is done using Volocity software (Ver. 5.0.0)
(see below).
m
