Sean Warren, who is a former member of the Photonics group in Physics and who wrote the FLIMfit software package for analysing fluorescence lifetime image data, will be giving a seminar on Monday 12th November from 1-2pm in Blackett room B636: Dr. Sean Warren Garvan Institute of Medical Research In vivo quantification of FRET biosensors using multispectral FLIM-FRET Intravital imaging of genetically expressed Förster resonance energy transfer (FRET) biosensors can provide unique insights into the function of biological processes in a native context. However, quantitative analysis of intravital FRET data is often compromised by motion, tissue or drug autofluorescence and scattering. We demonstrate tools to overcome these challenges and so reliably obtain accurate FRET quantification in vivo. We first demonstrate a new open source software tool, Galene, for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that we can resolve intravital FRET images of intra-abdominal organs in a murine model. We then introduce a multispectral fluorescence lifetime imaging (FLIM)-FRET analysis approach which allows reliable quantification of FRET biosensors in a variety of intravital contexts. We demonstrate that we can measure FRET activity in the presence of high levels of autofluorescence from both tissue and administered small molecule drugs. Finally, we demonstrate that this multispectral approach can be used to image two highly overlapping FRET biosensors using a genetically engineered mouse model expressing FRET biosensors for the prototypical RhoGTPases RhoA and Rac1. This approach allows us to directly visualise the mutually inhibitory response of RhoA and Rac1 on a subcellular level to treatment with small molecule inhibitors and activators. Best wishes Andreas ---------------------------------------------------- Dr Andreas Bruckbauer FILM facility manager Sir Alexander Fleming Building South Kensington Campus Imperial College London Exhibition Road London, SW7 2AZ SAF 4th floor, open space desk 401 +44 (0)20 7594 2023 a.bruckbauer@imperial.ac.uk<mailto:a.bruckbauer@imperial.ac.uk> http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/ http://www.imperial.ac.uk/people/a.bruckbauer ----------------------------------------------------