Biomedical Engineering Reference
In-Depth Information
Fig. 5
FRAP of Cdc3G at hyphal tips of
U. maydis
(
a
) Fluorescence micrographs illustrating the chronology of
a FRAP experiment: fl uorescence recovery underlies endosomal transport of Cdc3G [
14
]; different steps and
selected time points are indicated (bar = 10
m). (
b
) Fluorescence recovery is plotted against time, half-time
recovery (t
1/2
,
vertical dashed line
), and mobile as well as immobile fractions (
double arrows
) are deduced
from the fi tted curve (
red
), and
horizontal dashed line
indicates plateau
ʼ
5. Before bleaching, take a
z
-stack of 10 planes with an exposure
time of 500 ms and a distance between single planes of 0.5
ʼ
m
(
see
Note 22
).
6. Bleach 15
m of the hyphal tip in ten
z
-planes through fungal
hyphae with a
z
-distance of 0.5
ʼ
m.
7. Immediately acquire fl uorescence recovery in ten
z
-planes with
an exposure time of 500 ms each with a
z
-distance of 0.5
ʼ
ʼ
m
(open camera shutter).
8. Take a
z
-stack every minute for a period of 65 min (Fig.
5a
;
see
Notes 22
and
23
).
9. For data analysis, convert all z-stacks to maximum projections
and correct for drift of cells during the experiment (
see
Note 24
).
10. Subtract average background in each image (
see
Note 25
).
11. Determine the amount of acquisition bleaching and correct all
images (
see
Note 26
).
12. Normalize postbleach intensities to prebleach intensities, plot
fl uorescence recovery against time, apply curve fi tting, and
determine half-time recovery as well as mobile and immobile
fraction of protein with Prism 5 software (GraphPad, Fig.
5b
).
3.6 PALM
Experiments
1. Inoculate spores of
A. nidulans
strain expressing mEosFP-
TeaR in a glass-bottom chamber with minimal media. Incubate
at 25 °C for 12 h.
2. With low 473 nm illumination, identify a cell that expresses a
high level of fl uorophores.
3. Adjust laser intensity to image mEosFP. Initially, illuminate the
sample with the 561 nm laser to bleach the small population of
mEosFP that is already converted, e.g., emits red fl uorescence
(
see
Note 27
).
