Biomedical Engineering Reference
In-Depth Information
dynamic range of the detector. Evaluate the sample and
image for potential light-induced movement of LD, for cell
destruction, and for pixels exceeding the dynamic range of
the detector. Increase laser power and optimize image
acquisition (
see
Notes 15
-
17
).
3. Acquire an image at 2,840 cm
−1
(CH
2
stretching vibrations).
4. Tune the CARS system to 2,105 cm
−1
to probe CD
2
stretching
vibrations of deuterium-labeled neutral lipids.
5. Acquire an image at 2,105 cm
−1
.
6. (Figure
6
: CARS images of deuterium-labeled and unlabeled
fatty acids incorporated into neutral lipids of yeast cells).
4
Notes
1. Media for yeast cultivation, such as complete media (YPD),
contain large quantities of fl uorescent metabolites, which may
cause signifi cant spurious fl uorescence signals especially at the
interface between the coverslip and the liquid phase. This phe-
nomenon may interfere with high-resolution imaging of fl uo-
rescently labeled yeast structures, and cells may have to be
washed prior to the staining procedure. However, to avoid sig-
nifi cant changes of the cellular physiology and morphology
during the staining procedure and washing steps (e.g., the for-
mation of large vacuoles), we prefer to stain the cells directly in
the respective cultivation media. Appropriate emission fi lter
settings (spectral detector) may help to eliminate background
fl uorescence emitted from the medium.
2. RediGrad™ density gradient centrifugation was previously
shown to be largely inert on yeast cell physiology [
31
]. The
resulting cell population is very homogeneous in terms of cell
size, labeling characteristics, and growth. If cells are grown for
up to 7 days in stationary phase prior to RediGrad™ enrich-
ment, they are arrested in G0 phase of the cell cycle; after
transferring to fresh media, cells rapidly and synchronously
enter the cell cycle [
15
].
3. After mounting the coverslip, the cells are spread out as a
monolayer in a liquid fi lm, which should not extend to the
horizontal borders of the coverslip, indicative of too much liq-
uid that results in fl oating of the cells. By cutting several verti-
cal and horizontal slits into the agar, a large number (up to 96)
of different small yeast colonies can be mounted on a single
agar sheet without mixing. This setup enables image-based
high-content screens [
33
,
39
].
4. For CARS imaging of yeast LD, we apply high-numerical-
aperture objectives such as a 63× NA1.4 oil-immersion objective
