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  • JOURNAL ARTICLE
    Watson, Andrews N, Davis S, Bugeon L, Dallman M, McGinty Jet al., 2017,

    OPTiM: optical projection tomography integrated microscope using open-source hardware and software

    , PLOS One, Vol: 12, ISSN: 1932-6203

    We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 μm). All three methods use the microscope’s existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations’ viability.

  • CONFERENCE PAPER
    Jha A, Progatzky F, Wane M, Thwaites RS, McBrien M, Brimley J, Tunstall T, Shattock RJ, Bugeon L, Openshaw PJM, Dallman MJ, Hansel TTet al., 2016,

    Human nasal mucosal responses to TLR agonists are mirrored by the zebrafish gill

    , British Association of Lung Research Summer Congress

    Introduction: There are few reliable ways to study respiratory mucosal immune responses to viruses, viral-type toll-like receptor (TLR) agonists and vaccines. To investigate innate immune responses to TLR agonists (TLR3: poly IC/ poly ICLC; TLR7/8: resiquimod), we compared the effects on human nasal mucosa and zebrafish gills in vivo. Methods: Nasal challenge of adult volunteers was performed with saline, poly IC (n=4), poly ICLC (n=4) or resiquimod (n=8; 5 non-atopic, 3 atopic). Nasal mucosal lining fluid (MLF) was obtained by nasosorption at regular intervals up to 24 hours after challenge; nasal obstruction was monitored by peak nasal inspiratory flow (PNIF) and total nasal symptom scores (TNSS). Cytokines and interferons were measured in MLF using electrochemiluminescence on the Meso Scale Discovery (MSD) platform. Adult zebrafish gills were exposed to the same TLR agonists and gene expression was quantified in gill tissue at similar time-points. Results: Nasal challenge with TLR3 agonists failed to elicit any significant responses when compared to saline. In contrast resiquimod (10μg/100μl per nostril) caused a potent induction of cytokines with an early release (1-3 hours) of IFN-α2a, TNF-α and IL-1β and a later release (after 4 hours) of IFN-γ. The 3 volunteers with the highest levels of IFN-α2a were atopic. Six volunteers were asymptomatic and two volunteers had flu-like symptoms. There were no significant changes in clinical correlates of nasal obstruction. After resiquimod administration, but not TLR3 agonists, zebrafish gills showed an immune profile remarkably analogous to human nasal responses. Conclusion: The TLR7/8 agonist resiquimod is a potent mucosal inducer of IFN-α2a, IFN-γ and proinflammatory cytokines, whilst TLR3 agonists failed to stimulate mucosal innate immune responses. Zebrafish gills accurately mimic human nasal mucosal responses following exposure to TLR agonists, offering translational app

  • JOURNAL ARTICLE
    Kumar S, Lockward N, Ramel M-C, Correia T, Ellis M, Alexandrov Y, Andrews N, Patel R, Bugeon L, Dallman M, Brandner S, Arridge S, Katan M, McGinty J, Frankel P, French PMWet al., 2016,

    Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish

    , Oncotarget, Vol: 7, Pages: 43939-43948, ISSN: 1949-2553

    We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.

  • JOURNAL ARTICLE
    French PMW, andrews N, dallman M, ramel MC, kumar S, alexandrov Y, kelly D, warren S, kerry L, bugeon L, Lockwood N, Frolov Aet al., 2016,

    Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

    , Journal of Biophotonics, Vol: 9, Pages: 414-424, ISSN: 1864-0648

    Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.

  • JOURNAL ARTICLE
    Progatzky F, Cook HT, Lamb JR, Bugeon L, Dallman MJet al., 2016,

    Mucosal inflammation at the respiratory interface: a zebrafish model

    , AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, Vol: 310, Pages: L551-L561, ISSN: 1040-0605
  • JOURNAL ARTICLE
    Jones PJM, Sim A, Taylor HB, Bugeon L, Dallman MJ, Pereira B, Stumpf MPH, Liepe Jet al., 2015,

    Inference of random walk models to describe leukocyte migration

    , Physical Biology, Vol: 12, ISSN: 1478-3975

    While the majority of cells in an organism are static and remain relatively immobile in their tissue, migrating cells occur commonly during developmental processes and are crucial for a functioning immune response. The mode of migration has been described in terms of various types of random walks. To understand the details of the migratory behaviour we rely on mathematical models and their calibration to experimental data. Here we propose an approximate Bayesian inference scheme to calibrate a class of random walk models characterized by a specific, parametric particle re-orientation mechanism to observed trajectory data. We elaborate the concept of transition matrices (TMs) to detect random walk patterns and determine a statistic to quantify these TM to make them applicable for inference schemes. We apply the developed pipeline to in vivo trajectory data of macrophages and neutrophils, extracted from zebrafish that had undergone tail transection. We find that macrophage and neutrophils exhibit very distinct biased persistent random walk patterns, where the strengths of the persistence and bias are spatio-temporally regulated. Furthermore, the movement of macrophages is far less persistent than that of neutrophils in response to wounding.

  • JOURNAL ARTICLE
    Correia T, Lockwood N, Kumar S, Yin J, Ramel M-C, Andrews N, Katan M, Bugeon L, Dallman MJ, McGinty J, Frankel P, French PMW, Arridge Set al., 2015,

    Accelerated optical projection tomography applied to in vivo imaging of zebrafish

    , PLOS One, Vol: 10, ISSN: 1932-6203

    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.

  • JOURNAL ARTICLE
    Broncel M, Serwa RA, Ciepla P, Krause E, Dallman MJ, Magee AI, Tate EWet al., 2015,

    Myristoylation profiling in human cells and zebrafish.

    , Data in Brief, Vol: 4, Pages: 379-383, ISSN: 2352-3409

    Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.

  • JOURNAL ARTICLE
    Broncel M, Serwa RA, Ciepla P, Krause E, Dallman MJ, Magee AI, Tate EWet al., 2015,

    Multifunctional Reagents for Quantitative Proteome-Wide Analysis of Protein Modification in Human Cells and Dynamic Profiling of Protein Lipidation During Vertebrate Development

    , Angewandte Chemie-International Edition, Vol: 54, Pages: 5948-5951, ISSN: 1521-3773

    Novel multifunctional reagents were applied incombination with a lipid probe for affinity enrichment ofmyristoylated proteins and direct detection of lipid-modifiedtryptic peptides by mass spectrometry. This method enableshigh-confidence identification of the myristoylated proteomeon an unprecedented scale in cell culture, and allowed the firstquantitative analysis of dynamic changes in protein lipidationduring vertebrate embryonic development.

  • JOURNAL ARTICLE
    Chen L, Alexandrov Y, Kumar S, Andrews N, Dallman MJ, French PMW, McGinty Jet al., 2015,

    Mesoscopic in vivo 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography

    , BIOMEDICAL OPTICS EXPRESS, Vol: 6, Pages: 1253-1261, ISSN: 2156-7085
  • JOURNAL ARTICLE
    Progatzky F, Sangha NJ, Yoshida N, McBrien M, Cheung J, Shia A, Scott J, Marchesi JR, Lamb JR, Bugeon L, Dallman MJet al., 2014,

    Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation

    , NATURE COMMUNICATIONS, Vol: 5, ISSN: 2041-1723
  • CONFERENCE PAPER
    Progatzky F, Sangha NJ, Yoshida N, McBrien M, Cheung J, Shia A, Scott J, Marchesi JR, Lamb JR, Bugeon L, Dallman MJet al., 2014,

    Intestinal inflammation induced by dietary cholesterol in Zebrafish

    , 9th European-Mucosal-Immunology-Group Meeting, Publisher: WILEY-BLACKWELL, Pages: 27-28, ISSN: 0019-2805
  • JOURNAL ARTICLE
    Chen L, Kumar S, Kelly D, Andrews N, Dallman MJ, French PMW, McGinty Jet al., 2014,

    Remote focal scanning optical projection tomography with an electrically tunable lens

    , BIOMEDICAL OPTICS EXPRESS, Vol: 5, Pages: 3367-3375, ISSN: 2156-7085
  • JOURNAL ARTICLE
    Ciepla P, Konitsiotis AD, Serwa RA, Masumoto N, Leong WP, Dallman MJ, Magee AI, Tate EWet al., 2014,

    New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish

    , Chemical Science, Vol: 5, Pages: 4249-4259, ISSN: 2041-6520

    Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in theprogression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requiresattachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidatedShh has been challenging due to the limited array of tools available, and the roles of theseposttranslational modifications are poorly understood. Herein, we describe the development andvalidation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable itsvisualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown tobe an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shhfrom signalling cells, formation of multimeric transport complexes and signalling. We have used thisprobe to determine the size of transport complexes of lipidated Shh in culture medium and expressionlevels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitativechemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy anddetection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probesprovide a set of novel and well-validated tools that can be used to investigate the role of lipidation onactivity of Shh, and potentially other members of the Hedgehog protein family

  • JOURNAL ARTICLE
    Progatzky F, Dallman MJ, Lo Celso C, 2013,

    From seeing to believing: labelling strategies for in vivo cell-tracking experiments

    , INTERFACE FOCUS, Vol: 3, ISSN: 2042-8898
  • JOURNAL ARTICLE
    Taylor HB, Liepe J, Barthen C, Bugeon L, Huvet M, Kirk PDW, Brown SB, Lamb JR, Stumpf MPH, Dallman MJet al., 2012,

    P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish

    , Immunology and Cell Biology, Vol: 91, Pages: 60-69, ISSN: 1440-1711

    The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used.

  • JOURNAL ARTICLE
    Le Friec G, Sheppard D, Whiteman P, Karsten CM, Shamoun SA-T, Laing A, Bugeon L, Dallman MJ, Melchionna T, Chillakuri C, Smith RA, Drouet C, Couzi L, Fremeaux-Bacchi V, Koehl J, Waddington SN, McDonnell JM, Baker A, Handford PA, Lea SM, Kemper Cet al., 2012,

    The CD46-Jagged1 interaction is critical for human T(H)1 immunity

    , Nature Immunology, Vol: 13, Pages: 1213-1221, ISSN: 1529-2908

    CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (TH1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4+ T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate TH1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients.

  • CONFERENCE PAPER
    Le Friec G, Shamoun S, Couzi L, Fremeaux-Bacchi V, Baker A, Bugeon L, Dallman M, Handford P, Lea S, Kemper Cet al., 2012,

    Jagged1 is a CD46 ligand and disturbance in CD46/Jagged 1 interaction leads to abnormal Th1 function and infections in humans

    , European Congress of Immunology, Publisher: WILEY-BLACKWELL, Pages: 8-8, ISSN: 0019-2805
  • JOURNAL ARTICLE
    Progatzky F, Taylor H, Bugeon L, Cassidy S, Radbruch A, Dallman MJ, Lamb JRet al., 2012,

    The role of Nfil3 in zebrafish hematopoiesis

    , DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol: 38, Pages: 187-192, ISSN: 0145-305X
  • JOURNAL ARTICLE
    Gentle ME, Rose A, Bugeon L, Dallman MJet al., 2012,

    Noncanonical Notch Signaling Modulates Cytokine Responses of Dendritic Cells to Inflammatory Stimuli

    , JOURNAL OF IMMUNOLOGY, Vol: 189, Pages: 1274-1284, ISSN: 0022-1767
  • JOURNAL ARTICLE
    Rose A, Kay E, Wren BW, Dallman MJet al., 2012,

    The Campylobacter jejuni NCTC11168 capsule prevents excessive cytokine production by dendritic cells

    , MEDICAL MICROBIOLOGY AND IMMUNOLOGY, Vol: 201, Pages: 137-144, ISSN: 0300-8584
  • JOURNAL ARTICLE
    Chen L, McGinty J, Taylor HB, Bugeon L, Lamb JR, Dallman MJ, French PMWet al., 2012,

    Incorporation of an experimentally determined MTF for spatial frequency filtering and deconvolution during optical projection tomography reconstruction

    , OPTICS EXPRESS, Vol: 20, Pages: 7323-7337, ISSN: 1094-4087
  • JOURNAL ARTICLE
    Liepe J, Taylor H, Barnes CP, Huvet M, Bugeon L, Thorne T, Lamb JR, Dallman MJ, Stumpf MPHet al., 2012,

    Calibrating spatio-temporal models of leukocyte dynamics against in vivo live-imaging data using approximate Bayesian computation

    , INTEGRATIVE BIOLOGY, Vol: 4, Pages: 335-345, ISSN: 1757-9694
  • JOURNAL ARTICLE
    Bugeon L, Taylor HB, Progatzky F, Lin MI, Ellis CD, Welsh N, Smith E, Vargesson N, Gray C, Renshaw SA, Chico TJA, Zon LI, Lamb J, Dallman MJet al., 2011,

    The NOTCH pathway contributes to cell fate decision in myelopoiesis

    , HAEMATOLOGICA-THE HEMATOLOGY JOURNAL, Vol: 96, Pages: 1753-1760, ISSN: 0390-6078
  • JOURNAL ARTICLE
    Silk D, Kirk PDW, Barnes CP, Toni T, Rose A, Moon S, Dallman MJ, Stumpf MPHet al., 2011,

    Designing attractive models via automated identification of chaotic and oscillatory dynamical regimes

    , NATURE COMMUNICATIONS, Vol: 2, ISSN: 2041-1723
  • JOURNAL ARTICLE
    Lara R, Mauri FA, Taylor H, Derua R, Shia A, Gray C, Nicols A, Shiner RJ, Schofield E, Bates PA, Waelkens E, Dallman M, Lamb J, Zicha D, Downward J, Seckl MJ, Pardo OEet al., 2011,

    An siRNA screen identifies RSK1 as a key modulator of lung cancer metastasis

    , ONCOGENE, Vol: 30, Pages: 3513-3521, ISSN: 0950-9232
  • JOURNAL ARTICLE
    Lara R, Mauri F, Taylor H, Derua R, Shia A, Gray C, Nicols A, Shiner RJ, Schofield EE, Bates P, Waelkens E, Dallman M, Lamb J, Zicha D, Downward J, Seckl M, Pardo Oet al., 2011,

    IDENTIFICATION OF RSK1 AS A KEY MODULATOR OF LUNG CANCER METASTASIS

    , JOURNAL OF THORACIC ONCOLOGY, Vol: 6, Pages: S514-S515, ISSN: 1556-0864
  • JOURNAL ARTICLE
    Pridgeon C, Bugeon L, Donnelly L, Straschil U, Tudhope SJ, Fenwick P, Lamb JR, Barnes PJ, Dallman MJet al., 2011,

    Regulation of IL-17 in chronic inflammation in the human lung.

    , Clin Sci (Lond), Vol: 120, Pages: 515-524

    The regulation of human Th17 cell effector function by Treg cells (regulatory T-cells) is poorly understood. In the present study, we report that human Treg (CD4(+)CD25(+)) cells inhibit the proliferative response of Th17 cells but not their capacity to secrete IL (interleukin)-17. However, they could inhibit proliferation and cytokine production by Th1 and Th2 cells as determined by IFN-γ (interferon-γ) and IL-5 biosynthesis. Currently, as there is interest in the role of IL-17-producing cells and Treg cells in chronic inflammatory diseases in humans, we investigated the presence of CD4(+)CD25(+) T-cells and IL-17 in inflammation in the human lung. Transcripts for IL-17 were expressed in mononuclear cells and purified T-cells from lung tissue of patients with chronic pulmonary inflammation and, when activated, these cells secrete soluble protein. The T-cell-specific transcription factors RORCv2 (retinoic acid-related orphan receptor Cv2; for Th17) and FOXP3 (forkhead box P3; for Treg cells) were enriched in the T-cell fraction of lung mononuclear cells. Retrospective stratification of the patient cohort into those with COPD (chronic obstructive pulmonary disease) and non-COPD lung disease revealed no difference in the expression of IL-17 and IL-23 receptor between the groups. We observed that CD4(+)CD25(+) T-cells were present in comparable numbers in COPD and non-COPD lung tissue and with no correlation between the presence of CD4(+)CD25(+) T-cells and IL-17-producing cells. These results suggest that IL-17-expressing cells are present in chronically inflamed lung tissue, but there is no evidence to support this is due to the recruitment or expansion of Treg cells.

  • JOURNAL ARTICLE
    McGinty J, Taylor HB, Chen L, Bugeon L, Lamb JR, Dallman MJ, French PMWet al., 2011,

    In vivo fluorescence lifetime optical projection tomography

    , Biomedical Optics Express, Vol: 2, Pages: 1340-1350, ISSN: 2156-7085

    We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.

  • JOURNAL ARTICLE
    Kumar S, Alibhai D, Margineanu A, Laine R, Kennedy G, McGinty J, Warren S, Kelly D, Alexandrov Y, Munro I, Talbot C, Stuckey DW, Kimberly C, Viellerobe B, Lacombe F, Lam EW-F, Taylor H, Dallman MJ, Stamp G, Murray EJ, Stuhmeier F, Sardini A, Katan M, Elson DS, Neil MAA, Dunsby C, French PMWet al., 2011,

    FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ

    , CHEMPHYSCHEM, Vol: 12, Pages: 609-626, ISSN: 1439-4235

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