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| howto:flim-fret_calculation_for_single_exponential_donors [2014/06/18 09:16] – admin | howto:flim-fret_calculation_for_single_exponential_donors [2014/06/18 09:16] – admin |
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| This tutorial shows step-by-step, how the "Lifetime FRET Image" analysis of [[products:SymPhoTime64|SymPhoTime 64]] can be used to calculate pixel-by-pixel the FRET efficiency in an image containing a cell transfected with a [[wp>Green_fluorescent_protein|GFP]] and a RFP construct, that bind in certain regions of the cytoplasm. A bi-exponential fit is performed in a larger region of interest to determine the donor only lifetime and the lifetime of the donor in presence of [[glossary:fret|FRET]]. A pixel-by-pixel fit is performed in the image and in every pixel the FRET efficiency as well as the fraction of FRET on the total decay ("Binding") is determined. | This tutorial shows step-by-step, how the "Lifetime FRET Image" analysis of [[products:SymPhoTime64|SymPhoTime 64]] can be used to calculate pixel-by-pixel the FRET efficiency in an image containing a cell transfected with a [[wp>Green_fluorescent_protein|GFP]] and a RFP construct, that bind in certain regions of the cytoplasm. A bi-exponential fit is performed in a larger region of interest to determine the donor only lifetime and the lifetime of the donor in presence of [[glossary:fret|FRET]]. A pixel-by-pixel fit is performed in the image and in every pixel the FRET efficiency as well as the fraction of FRET on the total decay ("Binding") is determined. |
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| {{ youtube>Ka51hZNw3u8?Large }} | {{ youtube>Ka51hZNw3u8?large }} |
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| ===== Background information ===== | ===== Background information ===== |
