Prof Paul French, Imperial College London
Following a HEFCE JIF(V) award and funding from BBSRC, DTI/TSB (Beacon Award/Technology Award) EPSRC and the Wellcome Trust, the Photonics Group at Imperial have established capabilities in multidimensional fluorescence imaging (MDFI) and metrology with a strong emphasis on fluorescence lifetime imaging (FLIM). This includes wide-field, confocal and multiphoton microscopes providing FLIM, polarisation-resolved and hyperspectral imaging, as well as automated multiwell plate readers, endoscopes, macroscopes, tomographic imaging systems and multidimensional fluorometers for single point measurements in cuvettes or via optical fibre probes. All of these instruments are developed in-house and we write our own data acquisition and analysis software tools. This permits us to undertake experiments not possible with commercially available instrumentation and we work closely with life scientists to explore and develop new interdisciplinary scientific opportunities. Previously we have applied MDFI to study signalling processes at the immune synapse of NK cells and in signalling networks associated with cancer (e.g. Rac, Ras and their effectors) for which we have developed multiplexed FRET readouts. For cell biology we have also studied lipid order in cell membranes and membrane nanotubes. We have also applied FLIM to tissue autofluorescence for the detection and study of disease in animal models and in patients. Currently we are developing technology to advance MDFI at the important bioimaging frontiers of super resolution, high throughput and higher physiological relevance. For super resolved microscopy, we demonstrated the first STED FLIM microscope, also demonstrating the first application of Ti:Sapphire-pumped supercontinuum excitation to STED microscopy and first reporting the use of time-gated detection to remove non-super-resolved signal components. We also incorporated electronically adjustable correction of aberrations using a spatial light modulator with which we are currently implementing 3-D super-resolved STED to study the immunological synapse between interacting cell conjugates. For high throughput applications we have developed automated FLIM multiwell plate readers incorporating optical sectioning, image segmentation and global analysis of FRET readouts for high content analysis (HCA) of protein-protein interactions and other biomolecular assays. For higher physiological relevance we have developed oblique plane microscopy (OPM), optical projection tomography (OPT) and diffuse fluorescence tomography (DFT) for studies of cell biology in live disease models, including FRET readouts of biomolecular interactions. We have also developed fibre-optic based multidimensional fluorimetry and intravital microscopy and endoscopy to study tissue autofluorescence and have extended this to in vivo studies in animal models and patients.
This multidimensional fluorescence imaging and metrology can entail recording image data sets resolved with respect to three spatial dimensions, elapsed time and one or more spectroscopic dimensions including excitation and emission wavelength, fluorescence lifetime and/or polarisation. A major challenge for this technology platform is data analysis and management. Manual analysis of MDFI time-lapse data (movies) is extremely time-consuming and tedious to the point of being practically impossible and so we are developing automated image segmentation and global analysis tools for MDFI, which in turn, are producing vast quantities of data to be archived and reviewed. As an example, a 96 well plate FLIM data set in raw form is >68 GBytes. There is no commercially available software to handle such MDFI data and no integrated open sources solution to date. We have developed world-class, sophisticated software tools for analysing FLIM data but have not had the resources to develop coherent strategy for processing or archiving this data – or for readily making it publicly available. OMERO represents a timely and powerful opportunity to address this challenge, which is faced by many researchers at Imperial besides our laboratory, and which would greatly enhance our research. For this project we implement our MDFI analysis software into OMERO and also provide a “help-desk” function across Imperial to roll-out this tool to a community of >1000 scientists undertaking image-based research. This will include development of new bespoke analysis tools to be implemented into OMERO.
Further information is available on the Imperial Photonics Group website
Professor Paul French, Head of Photonics Group, was a Royal Society University Research Fellow at Imperial from 1989 and joined the academic staff in 1994. He has been a Visiting Professor at the University of New Mexico and a Consultant at AT&T Bell Laboratories. His research has evolved from ultrafast dye and solid-state laser physics to biomedical optics with a particular emphasis on FLIM for applications in molecular cell biology, drug discovery and clinical diagnosis. His current portfolio includes the development and application of multidimensional fluorescence imaging for microscopy, endoscopy and tomography and multidimensional fluorometric instrumentation for cuvette studies and in situ measurements via fibre-optic probes. He is a Fellow of the Institute of Physics, the European Physical Society and the Optical Society of America and holds a Royal Society Wolfson Research Merit Award. He is a life member of the Holland Club.
Ian Munro was born in Nottingham and has worked in the Photonics group of Imperial College London for the last 20 years developing software for a variety of digital image processing systems in particular for time-critical applications. He is currently working in the field of Fluorescence Lifetime Imaging (FLIM) for a variety of applications in medicine and microscopy. He received his Phd in 2007, prior to which he spent over 10 years in industry as a software engineer. His interests include (or, more accurately, consist entirely of) rock-climbing.
Chris Dunsby is a joint lecturer between the Photonics Group, Department of Physics and the Division of Experimental Medicine in the Department of Medicine. He received his undergraduate degree in Physics from Bristol University in 2000 and then went on to do a PhD in the Photonics Group at Imperial. His research interests are centred on the application of photonics and ultrafast laser technology to biomedical imaging and include multiphoton microscopy, multiparameter fluorescence imaging and fluorescence lifetime imaging (FLIM). Current projects include the development of high throughput FLIM instrumentation for reading out protein-protein interactions via fluorescence resonance energy transfer, the development of fibre optic probe instruments to study the autofluorescence signature of diseased tissue in the gastrointestinal tract for diagnostic applications and the development of multispectral multiphoton FLIM instrumentation to study skin cancer. He has also developed a video-rate 3D fluorescence imaging technique called oblique plane microscopy, which utilises light-sheet illumination.
Yuriy Alexandrov joined the project with expertise in scientific software engineering and biomedical image and data analysis. Holding a PhD in Applied Physics from Hebrew University of Jerusalem, Yuriy has been working for imaging research companies such as GE Healthcare’s HTS/HCA division. His interests also include mathematical modelling of biosystems and measurements. Yuriy is presently working at Imperial College London in the Photonics Group, where he is involved in applications of OMERO with FLIM and related techniques.
Sean Warren is a joint PhD student at the Photonics Group, Imperial College London and the Department of Structural and Molecular Biology, University College London developing multiplexed FLIM-FRET methodologies to study cell signalling pathways in chemotaxis. He developed FLIMfit as part of his PhD project. He received his undergraduate degree in Physics from the University of Cambridge in 2008 and a masters degree in Chemical Biology of Health & Disease from Imperial College London in 2010. He has previous worked as an Engineer at Roke Manor Research.