FRET View

In this view you can examine the results of the FRET analysis. The left image is the analyzed FRET image, the right is the original raw data image. The data table displays the results of the image analysis and differs whether you use the Sensitized Emission or Acceptor Bleaching method to measure FRET efficiency. With the view option tabs you can draw analysis regions into the raw data image and change analysis parameters.

Data Table Sensitized Emission

The analysis with the Sensitized Emission methods (all three methods are analyzed) shows the following parameters in the data table:

Parameter		Description
Region		Displays the identification number of regions assigned for analysis. The region zero refers to the whole image.
Time		Displays the time point if a time series is analyzed.
D avg.		Displays the average intensity of the region in the donor channel. These values are influenced by the selected settings in the Settings tab. They can vary from the values of the same regions in the Histo view.
A avg.		Displays the average intensity of the region in the acceptor channel. These values are influenced by the selected settings in the Settings tab. They can vary from the values of the same regions in the Histo view.
F avg.		Displays the average intensity of the region in the FRET channel. These values are influenced by the selected settings in the Settings tab. They can vary from the values of the same regions in the Histo view.
FRETN (p)		Displays the result of the analysis with the Gordon method. The FRETN value is calculated for each pixel, afterwards the average for the selected region is determined.
Fc (p)		Displays the result of the analysis with the Youvan method. The Fc value is calculated for each pixel, afterwards the average for the selected region is determined.
N-FRET (p)		Displays the result of the analysis with the Xia method. The N-FRET value is calculated for each pixel, afterwards the average for the selected region is determined.

Data Table Acceptor Bleaching

The analysis with the Acceptor Bleaching method shows the following parameters in the data table:

Parameter		Description
Region		Displays the identification number of regions assigned for analysis. The region zero refers to the whole image.
FRET(p) Eff. %		Displays the FRET efficiency for each pixel. It is calculated and averaged for all pixels of the region. The averaged intensities of the region are used to calculate an averaged FRET efficiency for this region. Delta D/D Post * 100.
D Pre		Displays the average donor intensity of the region in the pre-bleach image.
D Post		Displays the average donor intensity of the region in the post-bleach image.
A Pre		Displays the average acceptor intensity of the region in the pre-bleach image.
A Post		Displays the average acceptor intensity of the region in the post-bleach image.
Delta D		Displays the change in donor intensity of the region before and after the bleach event. Delta D = D Post-D Pre.
Delta A		Displays the change in acceptor intensity of the region before and after the bleach event. Delta A = A Post- A Pre.

FRET Tab

Parameter		Description
Toolbar		Offers tools to draw regions of interest into the image. These regions are then used for analysis.
–	Select	Activates the selection mode that enables you to select the graphic elements in the image area.
–	Clone	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.
–	Draw Rectangle	Enables you to draw a rectangular region of interest (ROI) into the image.
–	Draw Circle	Enables you to draw a circle.
–	Draw Ellipse	Enables you to draw a elliptical region.
–	Draw Spline Contour	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.
–	Draw Polygon	Enables you to draw a polygonal region.

Parameter		Description
Method		Selects the analysis method. The FRET image is updated depending on the selected method. For Sensitized Emission, ZEN provides three different methods for data analysis. The data table displays the results of all three methods. Using the Acceptor Bleaching method is only possible with an input of a dual channel time series with a bleach event.
Save Image		Opens the FRET image as new image document.
ROI Table		Displays the list of regions that are drawn into the raw data image.
–	#	Displays the identification number of the region.
–	Type	Displays the type of the region.
–	Object	Activated: Defines the region as one that should be analyzed.
–	Background	Activated: Defines the region as background region.

Parameters Tab

Parameter		Description
Donor Ch		Defines the channel which contains the Donor signal.
Acceptor Ch		Defines the channel which contains the Acceptor signal.
FRET Ch		Only visible for Sensitized Emission. Defines the channel which contains the FRET signal.
Image After Bleach Event		Only visible for Acceptor Bleaching . Selects the bleach event that should be analyzed in case of repetitive bleaching. The post-bleach image number is updated accordingly.
Pre-bleach Image:		Only visible for Acceptor Bleaching . Indicates the image before the bleach event used for analysis.
Post-bleach Image:		Only visible for Acceptor Bleaching . Indicates the image after the bleach event used for analysis.
Analyze		Only visible for Sensitized Emission. Selects the signal that should be analyzed in the present image.
–	Donor	Analyzes the donor signal of the image.
–	Acceptor	Analyzes the acceptor signal of the image.
Donor coef. Fd/Dd		Only visible for Sensitized Emission. Shows the value of Donor signal detected in the FRET channel/Donor signal detected in the Donor channel from the analyzed present image.
Donor coef. Ad/Fd		Only visible for Sensitized Emission. Shows the value of Donor Signal detected in the Acceptor channel/Donor signal detected in the FRET channel.
Acceptor coef. Fa/Aa		Only visible for Sensitized Emission. Shows the value of Acceptor signal detected in the FRET channel/Acceptor signal detected in the Acceptor channel.
Acceptor coef. Da/Aa		Only visible for Sensitized Emission. Shows the value of Acceptor signal detected in the Donor channel/Acceptor signal detected in the Acceptor channel.
Acceptor coef. Da/Fa		Only visible for Sensitized Emission. Shows the value of Acceptor signal detected in the Donor channel/Acceptor signal detected in the FRET channel.
G		Only visible for Sensitized Emission. Sets the Gordon Factor which depends on the fluorophores used and which can be part of the Gordon and Xia analysis. Refer to the original publications for further details (Gordon et al., Biophys J 74, 2702 (1998), Xia and Liu, Biophys J 81, 2395 (2001).
Global Coefficients		Only visible for Sensitized Emission. Activated: Applies the current coefficient values to all open image documents in FRET View.
Drop down selection		Only visible for Sensitized Emission. Selects the parameter that should be saved or loaded (Acceptor or Donor).
Save		Only visible for Sensitized Emission. Opens a file browser to save the coefficients of Donor or Acceptor as .xml file.
Load		Only visible for Sensitized Emission. Opens a file browser to load the coefficients of Donor or Acceptor for the current analysis.

Threshold Tab

On this tab, you can manually set the threshold for image analysis using the slider or input fields. Alternatively, the threshold can be set by defining and enabling a background region in the FRET tab.

The values for the thresholds are either displayed as grey value levels (Raw Data) or Normalized data, which is normalized to the value 1.

If you have activated Subtract Background Using Thresholds on the Settings tab, the background values are subtracted before the image analysis is performed.

Parameter		Description
Donor		Defines the threshold for the analysis of the donor signal.
Acceptor		Defines the threshold for the analysis of the acceptor signal.
FRET		Only visible for Sensitized Emission. Defines the threshold for the analysis of the FRET signal.
All		Defines the threshold for the analysis of all signals.
Clear		Sets all threshold values to zero and deactivates the background region.