6. Calculate the recovery at the five last time points to ensure that
the duration of recording the recovery is sufficient to enable
measurement and identification of several components (see
Note 10). If the change in the fluorescence intensity of the
ROI during these time points is greater than 1% of initial
intensity, increase the time of recording. In this case, one
must ensure that acquisitional photobleaching remains low. If
the change determined is lower than 1%, calculate the recovery
at the previous five time points. Use the last time point, at
which the recovery of the final five time points remains below
1%, to determine the duration required to record the recovery.
3.4 Bleaching
and Acquisition
1. Select a region of the epidermis for the experiment, set up a zstack, and select the ROI (Fig. 2a–d).
2. Set up the following experiment sequence (according to specific software and microscope): a 4D z-stack and time series at
selected resolution, time interval, and duration with bleaching
activated just before the start of the second or third z-section.
3. For measuring the dynamics of E-cad-GFP in Drosophila
embryos, use 8–10 embryos with 2–3 measurements per
embryo. Although the recovery of individual bleach events is
likely to be noisy and subjected to fluctuations in intensities,
this size of the dataset enables one to obtain a good and stable
averaged recovery curve (Fig. 3).
3.5 Signal Intensity
Measurements
and Data Processing
1. Open the raw “.oib/.czi” files and use the grouped z-projector
plugin to compile each time point as an average intensity
projection (see Note 11): for E-cad-GFP average intensity
complied by 6 for each stack taken, yields 45 time points. We
use Fiji (https://fiji.sc) for this purpose.
2. Select a region in the center of a cell without bleached junctions
and measure intensity using a ROI of the same size as used for
the bleached spots across the time series (see Note 12, Fig. 2e).
3. Perform the same for a control region, which is a junction
between two cells whose E-cad was not bleached, and for the
bleached regions (see Note 13, Fig. 2e).
4. Subtract background and normalize fluorescence intensity of
the bleached region as following: I n ¼ (F n À BG n )/
(FC n À BG n ), where F n is intensity of the bleached ROI at
the time point n, FC n is intensity of the control unbleached
ROI of the same size at the plasma membrane at the time point
n, and BG n is background intensity, measured with the same
size ROI in cytoplasm at the time point n.
5. Calculate the relative recovery at each time point using the
following formula: R n ¼ (I n À I 1 )/(I 0 À I 1 ), where I n , I 1 ,
and I 0 are the normalized intensities of bleached ROI at time
FRAP to Study the Dynamics of Proteins in vivo
151
the duration of recording the recovery is sufficient to enable
measurement and identification of several components (see
Note 10). If the change in the fluorescence intensity of the
ROI during these time points is greater than 1% of initial
intensity, increase the time of recording. In this case, one
must ensure that acquisitional photobleaching remains low. If
the change determined is lower than 1%, calculate the recovery
at the previous five time points. Use the last time point, at
which the recovery of the final five time points remains below
1%, to determine the duration required to record the recovery.
3.4 Bleaching
and Acquisition
1. Select a region of the epidermis for the experiment, set up a zstack, and select the ROI (Fig. 2a–d).
2. Set up the following experiment sequence (according to specific software and microscope): a 4D z-stack and time series at
selected resolution, time interval, and duration with bleaching
activated just before the start of the second or third z-section.
3. For measuring the dynamics of E-cad-GFP in Drosophila
embryos, use 8–10 embryos with 2–3 measurements per
embryo. Although the recovery of individual bleach events is
likely to be noisy and subjected to fluctuations in intensities,
this size of the dataset enables one to obtain a good and stable
averaged recovery curve (Fig. 3).
3.5 Signal Intensity
Measurements
and Data Processing
1. Open the raw “.oib/.czi” files and use the grouped z-projector
plugin to compile each time point as an average intensity
projection (see Note 11): for E-cad-GFP average intensity
complied by 6 for each stack taken, yields 45 time points. We
use Fiji (https://fiji.sc) for this purpose.
2. Select a region in the center of a cell without bleached junctions
and measure intensity using a ROI of the same size as used for
the bleached spots across the time series (see Note 12, Fig. 2e).
3. Perform the same for a control region, which is a junction
between two cells whose E-cad was not bleached, and for the
bleached regions (see Note 13, Fig. 2e).
4. Subtract background and normalize fluorescence intensity of
the bleached region as following: I n ¼ (F n À BG n )/
(FC n À BG n ), where F n is intensity of the bleached ROI at
the time point n, FC n is intensity of the control unbleached
ROI of the same size at the plasma membrane at the time point
n, and BG n is background intensity, measured with the same
size ROI in cytoplasm at the time point n.
5. Calculate the relative recovery at each time point using the
following formula: R n ¼ (I n À I 1 )/(I 0 À I 1 ), where I n , I 1 ,
and I 0 are the normalized intensities of bleached ROI at time
FRAP to Study the Dynamics of Proteins in vivo
151