Biomedical Engineering Reference
In-Depth Information
fresh YPD in a 12-well microtiter plate. Inoculate 3 × 10
7
cells
(OD 1;
see
Note 11
) of the preculture into the pre-warmed
YPD/fatty acid medium.
3. Cultivate the cells for 16 h at 30 °C on a thermomixer (350 rpm).
3.7 Microscopy
1. Mount cells on standard microscope slides or on agarose-
coated slides.
2. Visualize cells using DIC optics. The condenser position needs
to be adjusted for cells mounted on agarose-covered slides,
due to the extended distance from the condenser lens, for opti-
mal results (Fig.
2
: differential interference contrast imaging of
quiescent cells).
3.7.1 Transmission Light
Microscopy: Differential
Interference Contrast
(DIC, Nomarski)
BODIPY 493/503 fl uorescence is excited at 488 nm (Argon laser)
and emission detected between 500 and 550 nm. Thus, the dye is
not compatible with simultaneous GFP detection (GFP
λ
ex
/
λ
em
488/500-550 nm); however, it is compatible with red fl uorescent
proteins, e.g., mCherry or dsRed [
37
,
38
] (Fig.
3
).
Nile Red fl uorescence can be excited both at 488 nm (Argon
laser) and 543 nm (HeNe laser). For specifi c lipid droplet staining,
Nile Red is preferentially excited at 488 nm and fl uorescence emis-
sion detected between 550 and 575 nm. For detection of mem-
branes in addition to lipid droplets, Nile Red is preferentially
excited at 543 nm, and emission is detected between 550 and
575 nm for lipid droplets and between 600 and 700 nm for lipid
droplets plus intracellular membranes. Due to the rapid saturation
3.7.2 Confocal Laser
Scanning Microscopy
of Fluorescently
Labeled LD
Fig. 2
Differential interference contrast image of yeast cells prepared by density
gradient centrifugation and subsequent cultivation in complete media for 90 min.
Note the lack of any damaged cells and the high level of synchrony of cell divi-
sion. Bar = 10
μ
m
