This module enables you to analyze time series acquisitions with bleach events to determine the half time of recovery/decrease of fluorescent signals. It supports mono or bi-exponential fit algorithms, including options for background correction and correction of imaging-induced photobleaching. You also have the possibility to evaluate grouped regions of interest. Additionally, you can determine the fading factor from a reference region (Ref.) from the present experiment or a control experiment and reuse it for subsequent experiments.
FRAP Efficiency Analysis
FRAP View
The FRAP view (FRAP = Fluorescence Recovery after Photobleaching) is only visible for a time series data set which includes a minimum of one bleach event.
Note: When acquiring Airyscan SR or Airyscan MPLX data, the FRAP view only works as expected if the complete time series date is processed.
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Diagram |
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Image |
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Data Table Optionally, you can have a table of the average intensity values of each region group (corrected for background and reference) for each time point and channel. |
FRAP Tab
Any changes performed with this tab are immediately updated in the FRAP view (diagram and table).
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Toolbar |
Offers tools to draw regions of interest into the image. These regions are then used for analysis. |
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Activates the selection mode that enables you to select the graphic elements in the image area. |
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Activates the cloning mode that you can use to create an identical copy of the last graphic element drawn in by simply clicking anywhere into the image area. To exit this mode, either switch back to the selection mode or press the ESC key. |
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Enables you to draw a rectangular region of interest (ROI) into the image. |
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Enables you to draw a circle. |
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Enables you to draw a elliptical region. |
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Enables you to draw a spline contour. You can either define the corner points by a series of clicks or you can trace a contour by keeping the left mouse key pressed. Close this contour by right-clicking. Corners are always rounded with this tool. |
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Enables you to draw a polygonal region. |
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Parameter |
Description |
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Fit Formula |
Selects the mathematical model (mono or double exponential model) for data fitting. |
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Diagram Line |
Selects the line thickness of the fit curve(S) in the diagram. |
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Normalize |
Activated: Displays normalized values. The table of the intensity values and the fit diagram are updated accordingly. |
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Table |
Activated: Displays an additional table which shows the intensity values (for each channel) per time point. The values are corrected based on the background and reference region, if applied. |
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Diagram Marker |
Selects the time point markers of the fit curve(s) in the fit diagram. |
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Time Unit |
Selects the time units of the fit diagram, the fit data table and the intensity table. |
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Fit Range |
Defines the data range for the fit algorithm. |
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Photofading Factor |
The photofading factor is either calculated from the current reference region(s) or loaded from a previous experiment. The input field shows the currently applied value. The photofading factor = reference(t)/reference(t=0) and then fitted by: intensity(t) = exp(-kappa * t). |
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Use |
Activated: Uses the photofading factor for the data fitting algorithm. In the input field, you can enter the photofading factor. |
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Load |
Opens a file browser to load the photofading factor from an .xml file. |
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Save |
Opens a file browser to save the current photofading factor as an .xml file. |
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Region Table |
Displays the regions drawn into the image. |
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Displays the ID of the region. |
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Type |
Displays the type of the region. |
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Group 1, 2, 3 |
Allows you to select more than one region for analysis and to group them. The mean intensity values for the combined regions are used as data input. |
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Background |
Activated: Defines the region of interest which represents the mean background intensity that should be used for data correction. The mean intensity value of the background region is subtracted from the data prior to fitting. |
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Ref. |
Activated: Defines the region of interest which represents the fluorescence intensity of a reference area that has not been bleached and has not been affected by the bleach event. The mean intensity within this region is used to correct the data at each time point for any bleaching artifact that occurred during the imaging process. For this the data is divided by [reference(t)/reference(t=0)]. |
Fit Model
The Fit Model applied provides the following data/parameter:
- The final signal intensity in the analyzed ROIs following recovery IE (of the fitted curve)
- The amplitude of the fitted curve (which equals the mobile fraction) I1 mobile fraction
- The proportion of the mobile fraction: F1 mobile fraction (%)
- The fitted parameter T1(s)
- The half time of recovery T1 half (s)
- The rate constant for the exchange of molecules between the bleached region and the surrounding area K1 (1/s)
- The part of the immobile fraction of the protein I delta immobile fraction
- The proportion of the immobile fraction: F1 immobile fraction (%)
A double exponential displays the mean of the fitted values for the two different mobile fractions as the fit curve. The following (additional) parameters are provided
- The amplitude of the two curves, displayed as one (which corresponds to each part of the mobile fractions) I1 and I2.
- The fitted parameters T1 (s) and T2 (s) for each mobile fraction.
- The rate constant for the exchange of molecules between the bleached region and the surrounding area K1 (1/S) and K2 (1/s) for each mobile fraction.
- The half time of recovery for each fraction T1 half (s) and T2 half (s)
The table displays the result of the data fit. The result can be copied to the clipboard (right mouse click) and directly pasted into excel or saved as text.
The calculation of the parameters is based on the bleached ROI(s) unless other ROIs or selected or the ROIs are moved.
The analysis is then part of the image and the type of analysis is displayed again when opening the FRAP view for the image.