FLIM on a wide field fluorescence microscope
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FLIM (Fluorescence Lifetime Imaging Microscopy) is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM.
Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modular structure. Lifetime images showing FRET in MCF7 cells with ErbB1-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg are shown.
Key wordsFLIM fluorescence FRET LED lifetime microscopy
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