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--- howto:calculate_ratiometric_fret-images [2014/05/23 07:41] – admin
+++ howto:calculate_ratiometric_fret-images [2014/06/18 09:24] – admin
@@ Line 11: / Line 11: @@
 This tutorial shows step-by-step, how the "FRET Image" script of [[products:SymPhoTime64|SymPhoTime 64]] can be used to calculate pixel-by-pixel the average [[glossary:fret|FRET]] efficiency in an image containing several cells transfected with Cerulean/YFP constructs. To calculate the average FRET efficiency, a ratiometric approach based on the intensities in the donor and the acceptor channel is used.
-**Note:** [[glossary:fret|FRET]] is a method to determine molecular interactions or conformational changes based on a suited donor/acceptor pair.\\
+{{ youtube>rOas2iDwUzY?large }}
+===== Background Information =====
+[[glossary:fret|FRET]] is a method to determine molecular interactions or conformational changes based on a suited donor/acceptor pair.\\
 Intensity FRET as used here can only be used to calculate average FRET efficiencies. In case of a mixed population, these populations can only be separated using a donor dye with a single exponential decay kinetic and analyzed with lifetime data. This is not the scope of this tutorial.\\
 The script usage is demonstrated on an image of several cells which have been transfected with CENP-B-Cerulean and YFP-CENP-A. Cerulean is a CFP variant an frequently used as a FRET donor. The CENP-complex consists on several proteins and is involved in mitosis. For further details on the sample, see S. Orthaus et al., Chembiochem. 2008, 9, 77-92, and the PicoQuant application note [[http://www.picoquant.com/images/uploads/page/
@@ Line 36: / Line 41: @@
 {{ calculate_ratiometric_fret-images_with_the_fret-image-script_Image_3.png?348 }}
-**Note:**Comment: the drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}
+**Note:** the drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}
   * Start the "FRET Image" script by clicking on "Start".
@@ Line 121: / Line 126: @@
     * In case one wants to analyze many images with the same FRET pair, it is possible to save the starting values for the fits by clicking on "Save Defaults" and store the starting values as a User Profile by entering the main menu ("Settings\Save User Profile as"). The software will need to restart to adapt the changes.
   * If you don't want to do anything more, just close the result window.