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that are more fluorescent at the membrane than the mock-transfected control cells,
but do not give rise to detector saturation in the image, are selected for FFS measure-
ments. For cross-correlation measurements, only cells are selected that produce both
the mTq2 and sYFP2 fusion proteins. The laser power is set not higher than
2.5 kW cm
2
for the 440 nm laser line and 2.0 kW cm
2
for the 514 nm laser line
to prevent photobleaching and cellular damage and reduce photophysical effects.
Photobleaching and probe saturation lead to significant distortions of the correlation
curves, which will compromise the analysis. An overview
xy
-image is obtained at the
equatorial plane of the cell such that the fluorescent plasma membrane is located in
the middle (
Fig. 11.2
). In order to make use of the highest scanning rate of the laser
beam, that is, along the
x
-axis, the membrane should be visible as a vertical stripe in
the image. A horizontal line of 16 or 32 pixels is selected for measurements, inter-
secting the plasma membrane at the center of this line. The zoom factor is adjusted to
obtain pixel sizes of approximately 90
90 nm, and the line is scanned 32,766 times
at a rate of 1.7 ms per line. This measurement is repeated three times to improve pho-
ton statistics.
Finally, the samples of interest are being measured. All raw fluorescence inten-
sity files are saved for processing. If desired, cross-correlation could be measured in a
negative control experiment, which consists of mutants of the two sample proteins
lacking the possibility to interact with each other, for example, due to point mutations
or domain deletions. Alternatively, HeLa cells are cotransfected with separate con-
structs encoding for membrane-targeted mTq2 or sYFP2.
11.2.4
Auto- and cross-correlation analysis
Each raw data set is converted into a single kymograph tif image using the in-house-
built Ptu converter software. Only photons are exported from the CFP detector that
arrived within the first 25 n after the 440 nm laser pulse at
t
¼
0 ns. For the YFP chan-
nel, only the photons between
t
50 ns are exported. The image is
loaded into Matlab and processed using in-house-written scripts according to the fol-
lowing protocol.
First, the data are corrected for movement of the plasma membrane along the
x
-axis during the acquisition. Thereto, a moving averaged box of 20 lines is fitted
to an equation describing a Gaussian profile summed with a step function to take into
account intracellular fluorescence. Each individual line is translated such that the
peak fluorescence intensity in the box, which corresponds to the fluorescent plasma
membrane, will be located at the center of the line. From this aligned carpet, the five
central pixels per line are summed to generate a single intensity value per line. When
the intensity data set contains large intensity spikes (
¼
25 ns and
t
¼
10 times the intensity standard
deviation around the mean intensity), significant signal drift, or photobleaching
(
>
20% intensity loss per minute), the data set is discarded for further analysis. Stable
sections of the two intensity traces, one for each detection channel, are auto- and
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