Christopher Dunsby

Imperial College London, Londinium, England, United Kingdom

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Publications (51)105.61 Total impact

  • [Show abstract] [Hide abstract]
    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;
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    ABSTRACT: Degradation of articular cartilage extracellular matrix (ECM) by proteolytic enzyme is the hallmark of arthritis that leads to joint destruction. Detection of early biochemical changes in cartilage before irreversible structural damages become apparent is highly desirable. Here we report that the autofluorescence decay profile of cartilage is significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A multidimensional fluorometer utilizing ultraviolet excitation at 355 nm or 375 nm coupled to a fibreoptic probe was developed for single point time-resolved AFL measurements of porcine articular cartilage explants treated with different proteinases. Degradation of cartilage matrix components by treating with bacterial collagenase, matrix metalloproteinase 1, or trypsin resulted in significant reduction of AFL of the cartilage in both a dose and time dependent manner. Differences in cartilage AFL were also confirmed by fluorescence lifetime imaging microscopy (FLIM). Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may be utilized for diagnosis of arthritis as well as monitoring the efficacy of anti-arthritic therapeutic agents.
    02/2014;
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    ABSTRACT: Fluorescence lifetime imaging (FLIM) has previously been shown to provide contrast between normal and diseased tissue. Here we present progress towards clinical and preclinical FLIM endoscopy of tissue autofluorescence, demonstrating a flexible wide-field endoscope that utilised a low average power blue picosecond laser diode excitation source and was able to acquire ∼mm-scale spatial maps of autofluorescence lifetimes from fresh ex vivo diseased human larynx biopsies in ∼8 seconds using an average excitation power of ∼0.5 mW at the specimen. To illustrate its potential for FLIM at higher acquisition rates, a higher power mode-locked frequency doubled Ti:Sapphire laser was used to demonstrate FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
    Journal of Biophotonics 02/2014; · 3.10 Impact Factor
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    ABSTRACT: We describe a multicore endoscope fibre with minimised group index variation between cores obtained at a V parameter of 3. A spun fibre design enables the effects of bending to be reduced.
    Workshop on Specialty Optical Fibers and their Applications; 08/2013
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    ABSTRACT: We describe a multicore endoscope fibre with minimised group index variation between cores that is obtained at a V parameter of 3. Tapering the fibre input enables us to achieve single-mode propagation.
    CLEO: Science and Innovations; 06/2013
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    ABSTRACT: We demonstrate the application of Fluorescence Lifetime Imaging (FLIM) to read out Förster resonant energy transfer (FRET) based biosensors for studying the spatio-temporal dynamics of signalling pathways in cells undergoing chemotaxis.
    Optical Molecular Probes, Imaging and Drug Delivery; 04/2013
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    ABSTRACT: We report the development and application of instrumentation to measure and image tissue autofluorescence lifetime for the study and diagnosis of disease including cancer and osteoarthritis.
    Optical Molecular Probes, Imaging and Drug Delivery; 04/2013
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    ABSTRACT: Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a non-invasive investigation of the skin with subcellular resolution. The aim of this retrospective observational ex vivo study, was to characterize melanoma both from a morphologic and a quantitative point of view, attaining an improvement in the diagnostic accuracy with respect to dermoscopy. In the training phase, thirty parameters, comprising both cytological descriptors and architectural aspects, were identified. The training set included 6 melanomas with a mean Breslow thickness±S.D. of 0.89±0.48 mm. In the test phase, these parameters were blindly evaluated on a test data set consisting of 25 melanomas, 50 nevi and 50 basal cell carcinomas. Melanomas in the test phase comprised 8 in situ lesions and had a mean thickness±S.D. of 0.77±1.2 mm. Moreover, quantitative FLIM data were calculated for special areas of interest. Melanoma was characterized by the presence of atypical short lifetime cells and architectural disorder, in contrast to nevi presenting typical cells and a regular histoarchitecture. Sensitivity and specificity values for melanoma diagnosis were 100% and 98%, respectively, whereas dermoscopy achieved the same sensitivity, but a lower specificity (82%). Mean fluorescence lifetime values of melanocytic cells did not vary between melanomas and nevi, but significantly differed from those referring to basal cell carcinoma enabling a differential diagnosis based on quantitative data. Data from prospective preoperative trials are needed to confirm if MPT/FLIM could increase diagnostic specificity and thus reduce unnecessary surgical excisions.
    PLoS ONE 01/2013; 8(7):e70682. · 3.53 Impact Factor
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    ABSTRACT: BACKGROUND: Multiphoton Laser Tomography (MPT) has developed as a non-invasive tool that allows real-time observation of the skin with subcellular resolution. MPT is readily combined with time resolved detectors to achieve fluorescence lifetime imaging (FLIM). The aim of our study was to identify morphologic MPT/FLIM descriptors of melanocytic nevi, referring to cellular and architectural features. METHODS: In the preliminary study, MPT/FLIM images referring to 16 ex vivo nevi were simultaneously evaluated by 3 observers for the identification of morphologic descriptors characteristic of melanocytic nevi. Proposed descriptors were discussed and the parameters referring to epidermal keratinocytes, epidermal melanocytes, dermo-epidermal junction, papillary dermis and overall architecture were selected. In the main study, the presence/absence of the specified criteria were blindly evaluated on a test set, comprising 102 ex vivo samples (51 melanocytic nevi, 51 miscellaneous skin lesions) by 2 observers. RESULTS: Twelve descriptors were identified: "short-lifetime cells in the stratum corneum", "melanin-containing keratinocytes", "dendritic cells", "small short-lifetime cells" in the upper and lower layers", "edged papillae", "non-edged papillae", "junctional nests of short-lifetime cells", "dermal cell clusters", "short-lifetime cells in the papilla", "monomorphic and regular histoarchitecture", "architectural disarray". CONCLUSION: Identified descriptors for benign melanocytic lesions proved sensitive and specific, enabling the differentiation between melanocytic nevi and non-melanocytic lesions.
    Skin Research and Technology 12/2012; · 1.41 Impact Factor
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    ABSTRACT: Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.
    Matrix biology: journal of the International Society for Matrix Biology 12/2012; · 3.56 Impact Factor
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    ABSTRACT: Cell chemotaxis, such as migration of fibroblasts towards growth factors during development and wound healing, requires precise spatial coordination of signalling events. Phosphoinositides and signalling enzymes involved in their generation and hydrolysis have been implicated in regulation of chemotaxis, however, the role and importance of specific components remain poorly understood. Here, we demonstrate that phospholipase Cε (PLCε) contributes to fibroblast chemotaxis towards platelet-derived growth factor (PDGF-BB). Using PLCe1 null fibroblasts we show that cells deficient in PLCε have greatly reduced directionality towards PDGF-BB without detrimental effect on their basal ability to migrate. Furthermore, we show that in intact fibroblasts signalling events, such as activation of Rac, are spatially compromised by the absence of PLCε that affects the ability of cells to enlarge their protrusions in the direction of the chemoattractant. By further application of live cell imaging and the use of FRET-based biosensors, we show that generation of Ins(1,4,5)P3 and recruitment of PLCε are most pronounced in protrusions responding to the PDGF-BB gradient. Furthermore, the phospholipase C activity of PLCε is critical for its role in chemotaxis, consistent with the importance of Ins(1,4,5)P3 generation and sustained calcium responses in this process. As PLCε has extensive signalling connectivity, using transgenic fibroblasts we ruled out its activation by direct binding to Ras or Rap GTPases and suggest instead new, unexpected links for PLCε in the context of chemotaxis.
    Journal of Cell Science 09/2012; · 5.88 Impact Factor
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    ABSTRACT: AIMS: The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). METHODS: The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. RESULTS: MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). CONCLUSION: According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view.
    Skin Research and Technology 09/2012; · 1.41 Impact Factor
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    ABSTRACT: Tissue elasticity is closely related to the velocity of shear waves within biologic tissue. Shear waves can be generated by an acoustic radiation force and tracked by, e.g., ultrasound or magnetic resonance imaging (MRI) measurements. This has been shown to be able to noninvasively map tissue elasticity in depth and has great potential in a wide range of clinical applications including cancer and cardiovascular diseases. In this study, a highly sensitive optical measurement technique is proposed as an alternative way to track shear waves generated by the acoustic radiation force. A charge coupled device (CCD) camera was used to capture diffuse photons from tissue mimicking phantoms illuminated by a laser source at 532 nm. CCD images were recorded at different delays after the transmission of an ultrasound burst and were processed to obtain the time of flight for the shear wave. A differential measurement scheme involving generation of shear waves at two different positions was used to improve the accuracy and spatial resolution of the system. The results from measurements on both homogeneous and heterogeneous phantoms were compared with measurements from other instruments and demonstrate the feasibility and accuracy of the technique for imaging and quantifying elasticity. The relative error in estimation of shear wave velocity can be as low as 3.3% with a spatial resolution of 2 mm, and increases to 8.8% with a spatial resolution of 1 mm for the medium stiffness phantom. The system is shown to be highly sensitive and is able to track shear waves propagating over several centimetres given the ultrasound excitation amplitude and the phantom material used in this study. It was also found that the reflection of shear waves from boundaries between regions with different elastic properties can cause significant bias in the estimation of elasticity, which also applies to other shear wave tracking techniques. This bias can be reduced at the expense of reduced spatial resolution.
    Ultrasound in medicine & biology 06/2012; 38(9):1637-45. · 2.46 Impact Factor
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    ABSTRACT: Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus 'other lesions' was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of 'other lesion' extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.
    Experimental Dermatology 06/2012; · 3.58 Impact Factor
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    ABSTRACT: Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.
    PLoS Biology 09/2011; 9(9):e1001152. · 12.69 Impact Factor
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    ABSTRACT: Ultrasound-mediated optical tomography (UOT) is a hybrid technique that is able to combine the high penetration depth and high spatial resolution of ultrasound imaging to overcome the limits imposed by optical scattering for deep tissue optical sensing and imaging. It has been proposed as a method to detect blood concentrations, oxygenation and metabolism at depth in tissue for the detection of vascularized tumours or the presence of absorbing or scattering contrast agents. In this paper, the basic principles of the method are outlined and methods for simulating the UOT signal are described. The main detection methods are then summarized with a discussion of the advantages and disadvantages of each. The recent focus on increasing the weak UOT signal through the use of the acoustic radiation force is explained, together with a summary of our results showing sensitivity to the mechanical shear stiffness and optical absorption properties of tissue-mimicking phantoms.
    Interface focus: a theme supplement of Journal of the Royal Society interface 08/2011; 1(4):632-48. · 2.21 Impact Factor
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    ABSTRACT: Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.
    Proc SPIE 05/2011;
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    ABSTRACT: Background/purpose: Multiphoton microscopy (MPM) enables the assessment of unstained living biological tissue with submicron resolution, whereas fluorescence lifetime imaging microscopy (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. The aim of this study was to compare the healthy skin of young individuals with that of older subjects, as well as to assess the skin at different body sites, by means of MPM and FLIM.Methods: Nineteen elderly patients were examined on the outer side of the forearm, whereas 30 young individuals were assessed on the dorsal and volar sides of the forearm and on the thigh.Results: Cell and nucleus diameters, cell density and FLIM vary according to the epidermal cell depth and the skin site. In elderly subjects, epidermal cells show morphologic alterations in shape and size, with smaller cell and nucleus diameters; the number of basal cells is decreased, whereas the mean fluorescence lifetimes at both the upper and the lower layers increase.Conclusion: This study provides quantitative and qualitative data on normal epidermis at different skin sites at different ages and represents a reference for the clinician attempting to understand the effectiveness of MPM and FLIM in discriminating diseased states of the skin from normal ones.
    Skin Research and Technology 04/2011; 17(3):295 - 303. · 1.41 Impact Factor
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    ABSTRACT: To aid the in vivo diagnosis of skin lesions, we present the design and implementation of a 4 channel FLIM detector and a hyperspectral imaging detector into a clinically licensed commercial two-photon tomograph. We have also implemented image segmentation algorithms to facilitate the automated processing of the large volumes of data produced. The first detector is based on multispectral time correlated single photon counting, providing four channel fluorescence lifetime images. The second detector is a prism-based CCD hyperspectral imager. These detectors provide the capability to extract the relative content and state of autofluorescence compounds present in biological tissue.
    Proc SPIE 02/2011;
  • Biophysical Journal - BIOPHYS J. 01/2011; 100(3).

Publication Stats

418 Citations
105.61 Total Impact Points

Institutions

  • 2003–2014
    • Imperial College London
      • Department of Physics
      Londinium, England, United Kingdom
  • 2011
    • Institute of Cancer Research
      Londinium, England, United Kingdom