A major component of the Photonics Group research portfolio concerns the application of photonics to the study of disease and the development of diagnostic tools and therapies.  These biophotonics projects particularly utilise fluorescence readouts and range across the scales from cuvette-based solution studies of molecular biology and super-resolved microscopy through high content analysis (HCA) and preclinical imaging to clinical studies of patients. 

For solution-based measurements of biomolecular interactions we have developed a novel automated multidimensional fluorometer that is able to analyse fluorescence signals with respect to excitation and emission wavelength, fluorescence lifetime and polarisation in a single experiment1.  This may be used for cuvette-based studies and we are currently upgrading it to automated multiwell plate operation.  It is also able to operate coupled to a fibre-optic probe for remote measurements, e.g. of biological tissue.  This capability is being applied to label-free studies of cancer, heart disease and osteoarthritis. 

A large part of our biophotonics research concerns fluorescence imaging of cells and biological tissue.  Our instrumentation includes conventional wide-field and laser scanning confocal/multiphoton microscopes, including total internal reflection fluorescence (TIRF) and tandem scanning (Nipkow disc) instruments. These microscopes are modified for fluorescence lifetime imaging (FLIM) and we also resolve fluorescence signals with respect to excitation and emission spectra and polarisation, realising a multidimensional fluorescence imaging (MDFI) technology platform for biomedical research.  In collaboration with our colleagues in biology and medicine, we routinely apply our MDFI technology to Förster resonant energy transfer (FRET) of protein-protein interactions to analyse cell signalling networks for drug discovery and fundamental studies of molecular disease mechanisms.

Moving forward, we aim to leverage our expertise in photonics technology to create new tools and opportunities for molecular cell biology and drug discovery.  This is an exciting time for biophotonics as traditional barriers to observation are being pushed back and life scientists can envisage learning about biomolecular processes with unprecedented detail, speed and physiological relevance.   Our vision is to provide higher content analysis at all scales of measurement ad to make these tools as widely available as possible by developing low cost instrumentation with open source software.


To better understand cell signalling processes, we are developing super-resolving fluorescence microscopes based on stimulated emission depletion (STED) microscopy and stochastically-switched single molecule localisation microscopes based on PALM and STORM.  Our STED microscope was the first super-resolved microscope to incorporate FLIM2 and is being applied to multi-label imaging of protein interactions at the immunological synapse.  Non-biological applications include the study of nitrogen defects in diamond.

To study cell signalling networks and mechanisms of disease, we have developed a range of high-speed optically sectioned FLIM microscopes with single photon3 and (multibeam) multiphoton4 excitation for rapid 3-D imaging of cell biology, particularly using FRET.  For drug discovery it is important that the sophisticated experiments undertaken in advanced microscopes can be scaled to higher throughput so that the action of many potential drug compounds can be tested in biological systems and genomic array techniques can be used to identify the roles of specific genes in cell signalling networks and data can be systematically acquired for systems biology.   To this end we are exploiting our high-speed optically section FLIM microscopes that permit us to read out, e.g. FRET at rates up to ~10 Hz. The figure above includes on the left hand side an optically sectioned FLIM image (5 s acquisition) reading out FRET between Ras and Raf proteins in the EGF signal pathway3.  We have also developed a rapid optically sectioned multiplexed FLIM microscope that can read out two independent FRET signals, e.g. from two different components of a cell signalling network5.

For higher throughput/high content analysis (HCA), we have developed automated optically sectioned FLIM multiwell plate readers, based on home-built prototypes5 and a modified commercial instrument (GE Healthcare INCell 1000) [6], that are able to read a 96 well plate in ~ 10 minutes, including all sample translation and focussing steps6,7.  Together with its associated analysis software, this technology makes FLIM a practical tool for high content analysis (HCA) including for live cell assays. We have recently demonstrated the application of this FLIM HCA technology to both heteroFRET and homoFRET readouts of the oligomerisation of HIV-gag protein, realising Z' > 0.5 for these assays8 and have subsequently applied the technology to label-free readouts of changes in cellular metabolism utilising FLIM of NAD(P)H autofluorescence9

For drug discovery and for fundamental biomedical research, it is increasingly imperative to translate cell-based assays to in vivo studies.  Accordingly, we are developing tomographic FLIM instruments based on optical projection tomography (OPT) for optically cleared/transparent samples such as mouse10 and zebrafish embryos11 and diffuse fluorescence lifetime tomography (DFT)12, e.g. for mouse imaging.  Thus the same FLIM FRET assays can be compared in solution, in cells and in vivo.

For clinical applications, FLIM and MDFI can be applied to autofluorescence to provide label-free molecular contrast in biological tissue, e.g.13,14.  We are investigating the potential of ex vivo and in vivo FLIM with a view to developing diagnostic tools.  To this end we are developing a range of FLIM endoscopes including a FLIM confocal endomicroscope15, wide-field FLIM endoscopes and single point fibre-optic multidimensional fluorescence probes to provide more detailed information on complex spectro-temporal autofluorescence signals.  We have also developed a multispectral FLIM detection system for the DermaInspect™ multiphoton tomography instrument, which we applied to ex vivo and in vivo imaging of human skin16,17 and are working on developing hand-held multiphoton imaging instrumentation with built-in axial motion compensation18.

Open source software

We are increasingly working towards making our capabilities openly available for other academic research groups to replicate and develop. To this end we are making use of open source software where practical and provide versions of our software tools for download and non-commercial use.

To access FLIMfit, our FLIM data analysis software, please click here.

To access our software tools and equipment resource for openFLIM-HCA, please click here.


1 H.B. Manning et al., “A compact, multidimensional spectrofluorometer exploiting supercontinuum generation”, J. Biophoton. 1, 494–505 (2008)
2 E. Auksorius et al., “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging”, Opt Lett 33, 113-1 15 (2008)
3 D. M. Grant et al., “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events”, Opt. Expr.15 15656 -15673 (2007)
4 S. Kumar et al., “Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging”, Opt. Expr.15 12548-12561 (2007)
5 D. M. Grant et al., “Multiplexed FRET to monitor multiple signalling events in live cells”, Biophysical Journal: Biophysical Letters 95 (2008) L69-L71
6 C. B. Talbot et al., “High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis”, J. Biophoton. 1, 514–521 (2008)
7 S. Kumar et al., FLIM FRET technology for drug discovery: automated multiwell plate high content analysis, multiplexed readouts and application in situ”, ChemPhysChem 12 (2011), 627-633
8 Alibhai et al., “Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation", J. Biophotonics 6 (2012) 398-408
9 Kelly et al., "An automated multiwell plate reading FLIM microscope for live cell autofluorescence lifetime assays", J. Innov. Opt. Health Sci.. 7 (2014) 1450025-15 pages
10 J McGinty et al., “Fluorescence Lifetime Optical Projection Tomography”, J Biophotonics 1, 390-394 (2008)
11 J McGinty et al., "In vivo fluorescence lifetime optical projection tomography", Biomed. Opt. Expr. 2 (2011) 1340-1350
12 J. McGinty, V. Y. Soloviev, K. B. Tahir, R. Laine, D. W. Stuckey, Joseph V. Hajnal, A. Sardini, P M.W. French, and S. R. Arridge, “3-D imaging of Förster resonance energy transfer in turbid media by tozographic fluorescence lifetime imaging”, Opt. Lett. 34, 2272-2274 (2009)
13 C. B. Talbot et al., Fluorescence lifetime imaging and metrology for biomedicine, Chapter in Handbook of Photonics for Biomedical Science, Ed. Valery Tuchin, Published by Taylor and Francis 2010
14 J. McGinty et al., "Wide-field fluorescence lifetime imaging of cancer", Biomedical Optics Express, 1 (2010) 627-640
15 G.T. Kennedy et al., “A Fluorescence Lifetime Imaging Scanning Confocal Endomicroscope”, J.  Biophoton, 3, 103-107 (2010)
16 R. Patalay et al., "Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels", Biomedical Optics Express 2 (2012) 3295-3308
17 R. Patalay et al., "Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas", PLoS ONE 7 (9) (2012) e43460
18 B. Sherlock, S. Warren, J. Stone, M. Neil, C. Paterson, J. Knight, P. French and C. Dunsby. “Fibre-coupled multiphoton microscope with adaptive motion compensation”, Biomedical Optics Express 6 (5) (2015) 1876-1884