Biology Reference
In-Depth Information
Application of FRET and Microscopy
images had to be recorded with adapted laser intensities and exposure times. Wide-
field mode was measured with 30% laser intensity and 50 ms exposure time, and
TIRF did require 100% intensity and 500 ms exposure time.
14.3.6
Procedure part II: data evaluation
To determine apparent FRET efficiency for 5-HT
7
and the 5-HT
1A
receptor
interaction, we used lux-FRET method described in theory section. The detailed
data evaluation steps are illustrated in
Fig. 14.6
. For the imaging systems, various
additional steps are required, since evaluation is pixel-based. In the following, the
procedure steps 2-4, 6-8, 14, and 15 are only required for imaging systems.
FIGURE 14.6
Flowchart describing preprocessing and offline data evaluation of lux-FRET quantification for
data sets obtained at different setups.
