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--- howto:flim_fret_calculation_for_multi_exponential_donors [2014/05/23 07:43] – admin
+++ howto:flim_fret_calculation_for_multi_exponential_donors [2014/06/18 10:40] – admin
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 {{tag>tutorial howto analysis FLIM imaging FRET SymPhoTime}}
-~~TOC~~
+~~NOTOC~~
 ====== FLIM FRET Calculation for Multi Exponential Donors ======
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 This tutorial shows step-by-step, how the "Lifetime FRET Image" analysis of 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, first the donor only lifetime is determined using cells that have only been transfected with the donor construct by performing a ROI-fit with the FLIM-analysis. The amplitude weighted average donor lifetime is used to calculate the average FRET-efficiency using the "Lifetime FRET Image" analysis.
-**Note:**\\
+{{ youtube>zHfGae_Pd8A?large }}
+===== Background information =====
 FLIM-FRET is a robust method to determine the FRET efficiency of a suited donor acceptor pair.
 If the FRET-donor molecule has a more exponential decay kinetic, the amplitude weighted average lifetime has to be used to calculate the average FRET efficiency.
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     * 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.