Biomedical Engineering Reference
In-Depth Information
or a 63× NA1.2 water-immersion objective. For 2D imaging of
yeast cells with optimized sampling rate, a line frequency of
400 Hz (512×512 pixels) and bidirectional scanning mode
are applied. Optionally, 8× or 16× line averaging is performed.
Slower scan speed may result in light-driven artifi cial move-
ment of LD or may lead to cell destruction. For 3D imaging of
yeast cells, an increased line frequency of 700 Hz (512 × 512
pixels) and bidirectional scanning mode are applied. With this
setup no visible cell damage was observed. Using this setup
small LD (<200 nm) can also be imaged multidimensionally [
36
].
However, in 4D experiments over extended periods of time,
signifi cantly impaired cell growth, most likely caused by cell
stress, has been observed, using CARS. Yeast LD can also be
imaged at video-rate using a resonant scanner [
24
]. However,
by using this fast scanning mode, imaging of smaller LD
(<200 nm) is limited.
5. To supply cells with nutrients during long-term microscopic
observation, the agarose (agar) can be dissolved in growth
media. Using an objective heater system, this preparation tech-
nique also enables cultivation and microscopic analysis of yeast
cells directly on the microscope table, over extended periods of
time under close-to-physiological conditions.
6. Nile Red, BODIPY 493/503, and LD540 are practically non-
fl uorescent in the aqueous environment. Thus, we typically do
not wash the cells after staining, and confocal imaging of cells
in the presence of excess dye in the media has no signifi cant
effect on the background signal. Brightly fl uorescing entities
are typically dead cells. Of note, all three dyes have a high affi nity
for neutral lipids (i.e., LDs), but they also label the more polar
phospholipids. Thus, especially in growing cells that contain
reduced levels of neutral lipids and LDs, a signifi cant staining
of subcellular membranes is observed. Also, the staining of
membrane accumulations in mutant cells lacking neutral lipids
[
20
] may lead to misinterpretations.
7. BODIPY 493/503, LD540, and particularly Nile Red are
actively pumped out from actively growing yeast cells by the
activity of pleiotropic drug resistance pumps [
33
,
40
]. This
effect can be partially compensated for by maintaining cells on
fl uorescently labeled agar sheets (
see
Subheading
3.4
) or by
chemical fi xation of cells (
see
also
Note 9
).
8. LD540 is not yet commercially available. It is synthesized
according to Patent No. EP 2284541 A1: Fluorescence-based
imaging and analysis of cells and cellular components using
lipophilic dyes with improved specifi city, spectral property,
and photostability. Patent owner: Christoph Tiehle, Max-
Planck-Gesellschaft zur Förderung der Wissenschaften e.V.,
Germany.
