Biomedical Engineering Reference
In-Depth Information
9. To overcome limitations of the imaging of both GFP- and Nile
Red-labeled stationary phase yeast cells, i.e., photobleaching of
Nile Red or PDR-mediated active export of Nile Red from
yeast cells, we apply this short fi xation protocol in combination
with sequential imaging. In the sequential scan mode, param-
eters such as laser intensity and emission wavelength are opti-
mized for both GFP and Nile Red individually and are then
applied to the double-labeled fi xed yeast samples automatically
and in a sequential order. Fixed cells can be mounted in the
presence of anti-fading reagents to reduce photobleaching.
10. The fi xation time depends on the type of GFP fusion and on
the growth stage of the cells and has to be determined experi-
mentally. In our lab, stationary phase wild-type cells are fi xed
for 2-5 min and exponentially growing yeast cells are fi xed for
a maximum time of 60 s. In general, fi xation times should be
as short as possible since longer fi xation times may infl uence
morphology of lipid droplets and GFP fl uorescence.
Stabilization during fi xation by sorbitol effi ciently supports
GFP integrity and maintains its fl uorescence, but may interfere
with vacuole morphology.
11. The cell density is critical for effi cient fatty acid uptake. Cell
densities should be kept low to increase the specifi c cellular
fatty acid uptake.
12. We observed rapid photobleaching of Nile Red when applying
optimized zoom settings required for high-resolution confocal
imaging. However, the rate of Nile Red photobleaching is
strongly dependent on the intensity of the illuminating laser
source. Setting the laser intensity of the 488 nm argon laser
line or the 543 nm HeNe laser line of the confocal microscope
system to ~1-5 % or 50 %, respectively, of the maximum out-
put power, signifi cantly reduces Nile Red photobleaching.
However, these values may need adjustment as the laser power
decreases over time. Due to the excellent fl uorescence proper-
ties of Nile Red, high-contrast images with optimum resolu-
tion are obtained even at very low excitation intensities that
also enable reliable multidimensional imaging of Nile Red-
labeled structures.
13. LD540 is compatible with simultaneous detection of
GFP. However, the dye shows cross emission (“fl uorescence
crosstalk”) with the GFP channel when applied in higher con-
centrations. The emission wavelength for GFP detection
should therefore be further restricted and set between 500
and 530 nm and/or the concentration of LD540 should
be reduced to the lowest possible amount. Notably, at higher
concentrations and upon intense light illumination,
LD540 may induce fusion of LD (Wolinski, unpublished
observations, [
22
]).
