Abstract

Multidimensional Fluorescence Imaging For High Content Analysis and Preclinical Applications

Paul French, Professor, Department of Physics, Imperial College London

We are developing multidimensional fluorescence imaging technology with a particular emphasis on fluorescence lifetime imaging (FLIM) across the scales to contrast different molecular species and to map variations in the local fluorophore molecular environment, particularly due to Forster resonant energy transfer (FRET) in order to assay protein interactions or read out genetically expressed FRET biosensors. For high content analysis (HCA) we are implementing automated time-gated FLIM for multiwell plate assays of protein interactions or cellular metabolism. We have applied FLIM FRET HCA to study signalling and disease mechanisms in 2-D and 3-D cell-based assays, including the intracellular measurement of KD to quantify protein interactions. For in vivo preclinical studies, we are combining optical projection tomography (OPT) with FLIM, particularly applied to live zebrafish - from larvae up to adults - and are developing FLIM endoscopy to read out FRET biosensors in murine disease models. We aim to implement our multidimensional fluorescence imaging technology using open source software tools for instrument control, data acquisition, analysis and management and to provide lists of equipment components to enable other users to replicate our instrumentation for application to their own biological questions. Our open source FLIM analysis software, FLIMfit which provides rapid global fitting capabilities and is available as an OMERO client and our FLIM HCA and OPT instrumentation are controlled by µManager.