Much of today’s knowledge of cell biology and disease mechanisms has been obtained through molecular biology techniques, such as chromatography and Western blotting, that identify specific proteins and their interactions through chemical interactions with cell extracts in solution phase, or other biochemical analysis techniques including  NMR and mass spectrometry. These techniques, however, do not yet routinely provide single cell sensitivity but rather yield an analysis of cell populations. They have typically been complemented by microscopy of cells cultured in thin layers – typically one cell thick – on coverslips.  Such samples are highly amenable to optical imaging with high resolution and have provided information about cellular structures. Solution phase assays with optical readouts have been implemented in multiwell plates for highly parallelised, high throughput experiments able to automatically measure millions of samples. This has been heavily exploited in drug discovery to screen large libraries of compounds to identify drug candidates that have the desired biochemical effect. More recently, high content analysis platforms have been developed to automatically image cells with high throughput and provide readouts based on cell phenotype that utilise subcellular resolution. There is also increasing interest in exploiting microfluidics technology to increase the sensitivity and reduce the cost of solution-phase assays, e.g. for point of care applications. Although most of our biophotonics development concerns imaging technology, we have also applied multidimensional fluorescence techniques to cuvette and microfluidic-based measurements in solution.

Increasingly, sophisticated fluorescence labelling techniques are being applied to cell microscopy to permit single cell analysis of protein interactions, e.g. using FRET, and spatiotemporal dynamics of cell signalling and other processes, are being studied in fixed and live cells. These can include metabolic changes, which can be probed using label-free readouts of intracellular autofluorescence. Advances are being driven by innovation in imaging techniques, in software tools and particularly in approaches to labelling samples. In the Photonics Group we work on a wide range of cell imaging techniques and typically apply these capabilities to biological questions in partnership with colleagues from the life sciences. In terms of technology development, we are particularly interested in super-resolved microscopy, in automated high content analysis and in the translation of cell biology readouts to preclinical and clinical studies.

The following are some specific topics or projects in cell biology that we have addressed as part of our mission to understand the mechanism of disease in order to further the development of therapies:

Specific Topics or Projects

Solution phase and microfluidic measurements of processes in cell biology

Time-resolved Fluorescence Imaging of Microfluidic-based Binding Interactions
Richard K. P. Benninger, Bjorn Önfelt, Oliver Hofmann, Daniel M. Davis, Mark A. A. Neil, Paul M. W. French and Andrew J. deMello
Angewandte Chemie International Edition, 46 (2007) 2228-2231

Three-dimensional molecular mapping in a microfluidic mixing device using fluorescence lifetime imaging
T. Robinson, P. Valluri, H. B. Manning, D. M. Owen, I. Munro, C. B. Talbot, C. Dunsby, J. F. Eccleston, G. S. Baldwin, M. A. A. Neil, A. J. de Mello and P. M. W. French
Opt Lett 33 (2008) 1887-1889

High-throughput Flow-through Single Copy Polymerase Chain Reaction in Picolitre Droplets on Chip, Y. Schaerli, R. C. Wootton, T. Robinson, V. Stein, J. McGinty, Yasemin Koç, C. Dunsby, M. A. A. Neil, P. M. W. French, A. J. deMello, C. Abell and F. Hollfelder
Analytical Chemistry 81 (2009) 302-306

Differential modes of DNA binding by Mismatch Uracil DNA Glycosylase from E. coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway, S. Grippon, Q. Zhao, T. Robinson, J. J. T. Marshall, R. J. O’Neill, H. Manning4, G. Kennedy4, C. Dunsby, M. A. A. Neil, S. E. Halford5, P. M. W. French and G. S. Baldwin, Nucleic Acids Research, 39 (2010) 2593-2603.

Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy, Tom Robinson, Prashant Valluri, Gordon Kennedy, Alessandro Sardini, Christopher Dunsby, Mark A. A. Neil, Geoff S. Baldwin, Paul M. W. French, and Andrew J. de Mello, Analytical Chemistry: 86 (2014) 10732-10740

Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCg1, Louis Perdios, Tom D. Bunney, Sean C. Warren, Christopher Dunsby, Paul M.W. French, Edward W. Tate, Matilda Katan, Advances in Biological Regulation (2015),

Imaging cell membrane structure

Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes, Richard K. P. Benninger; Björn Önfelt; Daniel M. Davis; Mark Neil and Paul French, Biophysical Journal, 88 (2005) 609

Fluorescence Lifetime Imaging Provides Enhanced Contrast when Imaging the Phase-Sensitive Dye di-4-ANEPPDHQ in Model Membranes and Live Cells, D. M. Owen, P. M. P. Lanigan, C. Dunsby, I. Munro, D. Grant, M. A. A. Neil, P. M. W. French, and A. I. Magee, Biophysical Journal 90 (2006) L80-L82.

Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria, Önfelt B., Nedvetzki S., Benninger R.K.P., Purbhoo, M.A., Sowinksi S., Hume, A.N., Seabra, M.C., Neil M.A.A., French P.M.W., Davis D.M, Journal of Immunology. 177 (2006) 8476-8483

Optical techniques for imaging membrane lipid microdomains in living cells, D. M. Owen, M. A. A. Neil, P. M. W. French, and A. I. Magee, Seminars in Cell & Developmental Biology 18, (2007) 591-598

Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of Natural Killer cell immune synapses, Richard K.P. Benninger, Bruno Vanherberghen, Stephen Young, Sabrina B. Taner.‡ Fiona J. Culley, Tim Schnyder, Mark A.A. Neil, Daniel Wüstner, Paul M.W. French, Daniel M. Davis, and Björn Önfelt, Biophysical Journal: Biophysical Letters 96 (2008) L13-L15

High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells, D. M. Owen, S. Kumar, S. Oddos, C. Dunsby, M. Neil, D. M. Davis, P. M.W. French, M. L. Dustin, A. I. Magee and M. Cebecauer, Molecular Membrane Biology 27, 178-189 (2010).

Imaging the immunological synapse

Imaging the construction of immunological synapses
Davis, D. M.; Carlin, L. M.; Eleme, K.; French, P. M. W.; McCann, F.; Phillips, D.; Siegel, J.; Suhling, K.; Taner, S.; Vanherberghen, B.; Webb, S.; Yanagi, K, Tissue Antigens 59 (2002) 3-4

Microclusters of inhibitory killer immunoglobulin–like receptor signalling at natural killer cell immunological synapses, B. Treanor, P. M.P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A.A. Neil, D. N. Burshtyn, P. M.W. French and D. M. Davis, J Cell Biology, 174 (2006) 153-161

High-speed, high-resolution imaging of intercellular immune synapses using optical tweezers, S. Oddos, C. Dunsby, M. A Purbhoo, A. Chauveau, D. M Owen, M. A A Neil, D. M Davis & P. M W French, Biophysical Journal: Biophysical Letters 95 (2008) L66-L68

Dynamics of sub-synaptic vesicles and surface microclusters at the immunological synapse, M. A. Purbhoo, H. B. Liu, S. Oddos, D. M. Owen, M. A. A. Neil, S. V. Pageon, P. M. W. French, C. E. Rudd, and D. M. Davis, Science Signaling 3 (2010), p. ra36. [DOI: 10.1126/scisignal.2000645]

Remodelling of Cortical Actin where Lytic Granules dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy, A C N Brown, S Oddos, I M Dobbie, J-M. Alakoskela, R M Parton, P Eissmann, M A A Neil, C. Dunsby, P. M W French, I Davis, and D M Davis, PLoS ONE Biology 2011 September; 9 (9) : e1001152.

Imaging intracellular signalling

High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events, D. Grant, J. McGinty, E.J. McGhee, T.D. Bunney, D.M. Owen, C.B. Talbot, W. Zhang, S. Kumar, I. Munro, P. Lanigan, G. Kennedy, C. Dunsby, A.I. Magee, P. Courtney, M. Katan, M.A.A. Neil & P.M.W. French, Opt. Expr.15 (2007) 15656 - 15673

Multiplexed FRET to monitor multiple signalling events in live cells, D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I Munro, C. Dunsby, M. A. A. Neil, M. Katan and P. M. W. French, Biophysical Journal: Biophysical Letters 95 (2008) L69-L71

Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase C gamma 2, K. L. Everett, A. Buehler, T.D. Bunney, A. Margineanu, R. W. Baxendale, P. Vatter, M. Retlich, C. Walliser, H. B. Manning, M. A. A. Neil, C. Dunsby, P. M. W. French, P. Gierschik and M. Katan, Molecular and Cellular Biology, 31 (2011) 1240-1251

Activity of phospholipase C epsilon contributes to chemotaxis of fibroblasts towards platelet-derived growth factor, M. Martins, S. Warren, C. Kimberley, A. Margineanu, P. Peschard, A. McCarthy, M. Yeo, C. J. Marshall, C. Dunsby, P. M. French and M. Katan, J Cell Sci. (2012) 125 (Pt 23):5758-69. doi: 10.1242/jcs.110007.

SUMOylation inhibits FOXM1 activity and delays mitotic transition, S S Myatt, M Kongsema, C W-Y Man, D J Kelly, A R Gomes, P Khongkow, U Karunarathna, S Zona, J K Langer, C W Dunsby, R C Coombes, P M French, J J Brosens and E W-F Lam, Oncogene 33 (2014), 4316–4329,; doi: 10.1038/onc.2013.546

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation, D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineanu, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A.A. Neil, C. Dunsby and P. M.W. French, J. Biophotonics 6 (2012) 398-408, doi: 10.1002/jbio.201200185.

Automated multiwell fluorescence lifetime imaging for Förster resonant energy transfer assays and High Content Analysis, D. J. Kelly, S. C. Warren, D. Alibhai, S. Kumar, Y. Alexandrov, I. Munro, A. Margineanu, J. McCormack, N. J. Welsh, R. A. Serwa, E. Thinon, M. Kongsema, J. McGinty, C. Talbot, E. J. Murray, F. Stuhmeier, M. A.A. Neil, E. W. Tate, V. M. M. Braga, E. W.-F. Lam, C. Dunsby and P. M.W. French, Anal. Methods, 7 (2015), 4071-4089, DOI: 10.1039/C5AY00244C

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.
Warren SC, Margineanu A, Katan M, Dunsby C, French PM, Int J Mol Sci, (2015) DOI 10.3390/ijms160714695