P. M. W. French

Imperial College London, Londinium, England, United Kingdom

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Publications (363)758.24 Total impact

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    ABSTRACT: In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases.
    Advances in Biological Regulation 09/2015; DOI:10.1016/j.jbior.2015.09.002
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    ABSTRACT: Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections-achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.
    PLoS ONE 08/2015; 10(8):e0136213. DOI:10.1371/journal.pone.0136213 · 3.23 Impact Factor
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    ABSTRACT: To measure depth-specific riboflavin concentrations in corneal stroma using two-photon fluorescence microscopy and compare commercially available transepithelial corneal collagen cross-linking (CXL) protocols. Transepithelial CXL riboflavin preparations-MedioCross TE, Ribocross TE, Paracel plus VibeX Xtra, and iontophoresis with Ricrolin+-were applied to the corneal surface of fresh postmortem rabbit eyes in accordance with manufacturers' recommendations for clinical use. Riboflavin 0.1% (VibeX Rapid) was applied after corneal epithelial debridement as a positive control. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35-μm thick were cut on a cryostat, mounted on a slide, and imaged by two-photon fluorescence microscopy. Mean (SD) concentrations were calculated from five globes tested for each protocol. Peak riboflavin concentration of 0.09% (±0.01) was observed within the most superficial stroma (stromal depth 0-10 μm) in positive controls (epithelium-off). At the same depth, peak stromal riboflavin concentrations for MedioCross TE, Ricrolin+, Paracel/Xtra, and Ribocross TE were 0.054% (±0.01), 0.031% (0.003), 0.021% (±0.001), and 0.015% (±0.004), respectively. At a depth of 300 μm (within the demarcation zone commonly seen after corneal cross-linking), the stromal concentration in epithelium-off positive controls was 0.075% (±0.006), while at the same depth MedioCross TE and Ricrolin+ achieved 0.018% (±0.006) and 0.016% (0.002), respectively. None of the remaining transepithelial protocols achieved concentrations above 0.005% at this same 300-μm depth. Overall, MedioCross TE was the best-performing transepithelial formulation. Corneal epithelium is a significant barrier to riboflavin absorption into the stroma. Existing commercial transepithelial CXL protocols achieve relatively low riboflavin concentrations in the anterior corneal stroma when compared to gold standard epithelium-off absorption. Reduced stromal riboflavin concentration may compromise the efficacy of riboflavin/ultraviolet corneal CXL.
    Investigative ophthalmology & visual science 07/2015; 56(8):5006-5011. DOI:10.1167/iovs.15-16903 · 3.40 Impact Factor
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    Sean C Warren · Anca Margineanu · Matilda Katan · Chris Dunsby · Paul M W French ·
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    ABSTRACT: Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.
    International Journal of Molecular Sciences 06/2015; 16(7):14695-14716. DOI:10.3390/ijms160714695 · 2.86 Impact Factor
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    ABSTRACT: A correction is proposed to the Delta function convolution method (DFCM) for fitting a multiexponential decay model to time-resolved fluorescence decay data using a monoexponential reference fluorophore. A theoretical analysis of the discretised DFCM multiexponential decay function shows the presence an extra exponential decay term with the same lifetime as the reference fluorophore that we denote as the residual reference component. This extra decay component arises as a result of the discretised convolution of one of the two terms in the modified model function required by the DFCM. The effect of the residual reference component becomes more pronounced when the fluorescence lifetime of the reference is longer than all of the individual components of the specimen under inspection and when the temporal sampling interval is not negligible compared to the quantity (τR −1 – τ−1)−1, where τR and τ are the fluorescence lifetimes of the reference and the specimen respectively. It is shown that the unwanted residual reference component results in systematic errors when fitting simulated data and that these errors are not present when the proposed correction is applied. The correction is also verified using real data obtained from experiment.
    Journal of Fluorescence 06/2015; 25(5). DOI:10.1007/s10895-015-1583-4 · 1.93 Impact Factor
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    ABSTRACT: To address the challenge of sample motion during in vivo imaging, we present a fibre-coupled multiphoton microscope with active axial motion compensation. The position of the sample surface is measured using optical coherence tomography and fed back to a piezo actuator that adjusts the axial location of the objective to compensate for sample motion. We characterise the system’s performance and demonstrate that it can compensate for axial sample velocities up to 700 µm/s. Finally we illustrate the impact of motion compensation when imaging multiphoton excited autofluorescence in ex vivo mouse skin.
    Biomedical Optics Express 05/2015; 6(5). DOI:10.1364/BOE.6.001876 · 3.65 Impact Factor
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    ABSTRACT: Fluorescence lifetime measurements can provide quantitative assays of the local fluorophore environment and can be applied to read out biomolecular interactions via Förster resonance energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can be automated for high content analysis (HCA) to map protein–protein interactions with applications in drug discovery, systems biology and basic research. The automated acquisition of FLIM data over 100's of fields of view provides statistical power to overcome noise in instrumentation and biological systems and thus exploit relatively small changes in mean lifetime to provide useful readouts that would not be practically achievable in manual microscopy experiments. We present here an automated HCA system with the ability to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential through exemplar applications of different FRET readouts.
    Analytical methods 05/2015; 7(10). DOI:10.1039/C5AY00244C · 1.82 Impact Factor
  • J. McGinty · L. Chen · S. Kumar · Y. Alexandrov · N. Andrews · D. Kelly · M.J. Dallman · P.M.W. French ·
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    ABSTRACT: Optical projection tomography is a 3-D imaging approach applicable to transparent samples and model organisms like zebrafish embryos. We present methods to improve the spatial resolution and realize 3-D cell tracking in OPT.
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    ABSTRACT: We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and optical projection tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 projection angles. We demonstrate the application of this technique to mapping the 3-D neutrophil migration pattern observed over ~25.5 minutes in a live 2 day post-fertilisation transgenic LysC:GFP zebrafish embryo following a tail wound.
    Biomedical Optics Express 04/2015; 6(4). DOI:10.1364/BOE.6.001253 · 3.65 Impact Factor
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    Investigative Ophthalmology &amp Visual Science 02/2015; 56(2):1191-2. DOI:10.1167/iovs.15-16457 · 3.40 Impact Factor
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    ABSTRACT: We investigate the potential of an instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to measure structural and metabolic changes in cardiac tissue in vivo in a 16 week post-myocardial infarction heart failure model in rats. In the scar region, we observed changes in the fluorescence signal that can be explained by increased collagen content, which is in good agreement with histology. In areas remote from the scar tissue, we measured changes in the fluorescence signal (p < 0.001) that cannot be explained by differences in collagen content and we attribute this to altered metabolism within the myocardium. A linear discriminant analysis algorithm was applied to the measurements to predict the tissue disease state. When we combine all measurements, our results reveal high diagnostic accuracy in the infarcted area (100%) and border zone (94.44%) as well as in remote regions from the scar (> 77%). Overall, our results demonstrate the potential of our instrument to characterize structural and metabolic changes in a failing heart in vivo without using exogenous labels.
    Biomedical Optics Express 02/2015; 6(2). DOI:10.1364/BOE.6.000324 · 3.65 Impact Factor

  • IEEE Journal of Selected Topics in Quantum Electronics 01/2015; DOI:10.1109/JSTQE.2015.2488283 · 2.83 Impact Factor
  • B. Dyer · J. Lagarto · P. French · N. Peters · C. Dunsby · A. Lyon ·

    Heart (British Cardiac Society) 12/2014; 100(Suppl 4):A13-A13. DOI:10.1136/heartjnl-2014-306916.38 · 5.60 Impact Factor
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    ABSTRACT: Type 2 diabetes (T2D) is characterised by beta cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired beta cell function. Whilst deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the beta cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in beta cells from the earliest expression of the Ins1 gene ( approximately E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcf7L2fl/fl::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and16 weeks respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcf7L2fl/fl::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 ( approximately 20%) and Glp1r ( approximately 40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca2+ increases, and connectivity between individual beta cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed approximately 30 % decrease in beta cell mass in pancreata from Tcf7L2fl/fl::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of beta cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.
    Human Molecular Genetics 10/2014; DOI:10.1093/hmg/ddu553 · 6.39 Impact Factor
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    ABSTRACT: We describe a remote focal scanning technique for optical projection tomography (OPT) implemented with an electrically tunable lens (ETL) that removes the need to scan the specimen or objective lens. Using a 4× objective lens the average spatial resolution is improved by ∼46% and the light collection efficiency by a factor of ∼6.76, thereby enabling increased acquisition speed and reduced light dose. This convenient implementation is particularly appropriate for lower magnifications and larger sample diameters where axial objective scanning would encounter problems with speed and stability.
    Biomedical Optics Express 10/2014; 5(10). DOI:10.1364/BOE.5.003367 · 3.65 Impact Factor
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    ABSTRACT: Uracil DNA glycosylase (UNG) plays a key role in DNA maintenance via base excision repair (BER). Its role is to bind to DNA, locate unwanted uracil and remove it via a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the "binding" and "base flipping" steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor both binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.
    Analytical Chemistry 09/2014; 86(21). DOI:10.1021/ac502732s · 5.64 Impact Factor
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    ABSTRACT: Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine.
    Journal of Innovative Optical Health Sciences 09/2014; 07(05):1450025. DOI:10.1142/S1793545814500254 · 0.93 Impact Factor
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    ABSTRACT: Fully differentiated pancreatic β cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess ("glucotoxicity") is implicated in this process, we sought here to identify the potential roles in β-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK). Highly β-cell-restricted deletion of each kinase in mice, using an Ins1-controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0-12-fold, E<0.01) increased the expression of subsets of hepatic (Alb, Iyd, Elovl2) and neuronal (Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamate signaling. These changes were partially recapitulated by the loss of AMPK, which also up-regulated β-cell "disallowed" genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8- to 3.4-fold (E<0.01). Correspondingly, targeted promoters were enriched for neuronal (Zfp206; P=1.3×10(-33)) and hypoxia-regulated (HIF1; P=2.5×10(-16)) transcription factors. In summary, LKB1 and AMPK, through only partly overlapping mechanisms, maintain β-cell identity by suppressing alternate pathways leading to neuronal, hepatic, and other characteristics. Selective targeting of these enzymes may provide a new approach to maintaining β-cell function in some forms of diabetes.-Kone, M., Pullen, T. J., Sun, G., Ibberson, M., Martinez-Sanchez, A., Sayers, S., Nguyen-Tu, M.-S., Kantor, C., Swisa, A., Dor, Y., Gorman, T., Ferrer, J., Thorens, B., Reimann, F., Gribble, F., McGinty, J. A., Chen, L., French, P. M., Birzele, F., Hildebrandt, T., Uphues, I., Rutter, G. A. LKB1 and AMPK differentially regulate pancreatic β-cell identity.
    The FASEB Journal 07/2014; 28(11). DOI:10.1096/fj.14-257667 · 5.04 Impact Factor
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    ABSTRACT: We image core-shell nanoparticles, consisting of a dye-doped silica core covered with a layer of gold, with a STED-FLIM microscope. Due to the field enhancement provided by the localised surface plasmon resonance of the gold shell, we demonstrate a reduction of the STED depletion power required to obtain resolution improvement by a factor of four. This validates the concept of nanoparticle-assisted STED (NP-STED), where hybrid dye-plasmonic nanoparticles are used as labels for STED in order to decrease the depletion powers required for sub-wavelength imaging.
    Nano Letters 07/2014; 14(8). DOI:10.1021/nl5014103 · 13.59 Impact Factor
  • Christopher Dunsby · Paul French ·

    Fluorescence Lifetime Spectroscopy and Imaging, 07/2014: pages 103-116; , ISBN: 978-1-4398-6167-7

Publication Stats

6k Citations
758.24 Total Impact Points


  • 1986-2015
    • Imperial College London
      • • Department of Physics
      • • Department of Bioengineering
      Londinium, England, United Kingdom
  • 2011
    • Institute of Cancer Research
      Londinium, England, United Kingdom
    • Dong-A University
      • Department of Medicine
      Tsau-liang-hai, Busan, South Korea
  • 2010
    • University of Bristol
      • School of Biochemistry
      Bristol, England, United Kingdom
  • 2009
    • University of Southern Denmark
      Odense, South Denmark, Denmark
  • 2002
    • Purdue University
      ウェストラファイエット, Indiana, United States
  • 2000
    • University of Oxford
      • Department of Engineering Science
      Oxford, England, United Kingdom
  • 1997-1998
    • Imperial Valley College
      • Physics Department
      Imperial, California, United States