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ABSTRACT: We describe an angular multiplexing technique for optical projection tomography that improves resolution, signal-to-noise ratio, and imaging speed by ameliorating the trade-off between spatial resolution and depth of field and improving the light collection efficiency. Here we demonstrate that imaging at two orthogonal angular projections simultaneously, focused on shifted planes in the sample, improves the average spatial resolution by ∼20% and the light collection efficiency by a factor of ∼4, thereby enabling increased acquisition speed and reduced light dose.
Optics Letters 03/2013; 38(6):851-3. · 3.40 Impact Factor
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Dominic Alibhai,
Douglas J Kelly,
Sean Warren,
Sunil Kumar,
Anca Margineau,
Remigiusz A Serwa,
Emmanuelle Thinon,
Yuriy Alexandrov,
Edward J Murray,
Frank Stuhmeier,
Edward W Tate,
Mark A A Neil,
Chris Dunsby, Paul M W French
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ABSTRACT: Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ' factors exceeding 0.6 are realised for this FLIM FRET assay. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
Journal of Biophotonics 11/2012; · 4.34 Impact Factor
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Marta Martins,
Sean Warren,
Christopher Kimberley,
Anca Margineanu,
Pascal Peschard,
Afshan McCarthy,
Maggie Yeo,
Christopher J Marshall,
Christopher Dunsby, Paul M W French,
Matilda Katan
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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; · 6.11 Impact Factor
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ABSTRACT: We demonstrate two techniques to improve the quality of reconstructed optical projection tomography (OPT) images using the modulation transfer function (MTF) as a function of defocus experimentally determined from tilted knife-edge measurements. The first employs a 2-D binary filter based on the MTF frequency cut-off as an additional filter during back-projection reconstruction that restricts the high frequency information to the region around the focal plane and progressively decreases the spatial frequency bandwidth with defocus. This helps to suppress "streak" artifacts in OPT data acquired at reduced angular sampling, thereby facilitating faster OPT acquisitions. This method is shown to reduce the average background by approximately 72% for an NA of 0.09 and by approximately 38% for an NA of 0.07 compared to standard filtered back-projection. As a biological illustration, a Fli:GFP transgenic zebrafish embryo (3 days post-fertilisation) was imaged to demonstrate the improved imaging speed (a quarter of the acquisition time). The second method uses the MTF to produce an appropriate deconvolution filter that can be used to correct for the spatial frequency modulation applied by the imaging system.
Optics Express 03/2012; 20(7):7323-37. · 3.59 Impact Factor
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Alex J Thompson,
Sergio Coda,
Mikkel Brydegaard Sørensen,
Gordon Kennedy,
Rakesh Patalay,
Ulrika Waitong-Brämming,
Pieter A A De Beule,
Mark A A Neil,
Stefan Andersson-Engels,
Niels Bendsøe, Paul M W French,
Katarina Svanberg,
Chris Dunsby
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ABSTRACT: We present a clinical investigation of diffuse reflectance and time-resolved autofluorescence spectra of skin cancer with an emphasis on basal cell carcinoma. A total of 25 patients were measured using a compact steady-state diffuse reflectance/fluorescence spectrometer and a fibre-optic-coupled multispectral time-resolved spectrofluorometer. Measurements were performed in vivo prior to surgical excision of the investigated region. Singular value decomposition was used to reduce the dimensionality of steady state diffuse reflectance and fluorescence spectra. Linear discriminant analysis was then applied to the measurements of basal cell carcinomas (BCCs) and used to predict the tissue disease state with a leave-one-out methodology. This approach was able to correctly diagnose 87% of the BCCs. With 445 nm excitation a decrease in the spectrally averaged fluorescence lifetime was observed between normal tissue and BCC lesions with a mean value of 886 ps. Furthermore, the fluorescence lifetime for BCCs was lower than that of the surrounding healthy tissue in all cases and statistical analysis of the data revealed that this decrease was significant (p = 0.002).
Journal of Biophotonics 03/2012; 5(3):240-54. · 4.34 Impact Factor
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ABSTRACT: Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.
Dermatology Research and Practice 01/2012; 2012:810749.
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Rakesh Patalay,
Clifford Talbot,
Yuriy Alexandrov,
Martin O Lenz,
Sunil Kumar,
Sean Warren,
Ian Munro,
Mark A A Neil,
Karsten König, Paul M W French,
Anthony Chu,
Gordon W H Stamp,
Chris Dunsby
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ABSTRACT: We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm(2) is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425-515 nm spectral emission) to 39.8% (620-655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.
PLoS ONE 01/2012; 7(9):e43460. · 4.09 Impact Factor
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ABSTRACT: We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.
PLoS ONE 01/2012; 7(11):e49200. · 4.09 Impact Factor
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ABSTRACT: We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisition. Reconstructed image data are presented and compared with results obtained by using the telegraph equation approximation. The new approach provides improved recovery of absorption and scattering parameters while returning similar values for the fluorescence parameters.
Applied Optics 12/2011; 50(36):6583-90. · 1.41 Impact Factor
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ABSTRACT: We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed.
Biomedical Optics Express 12/2011; 2(12):3295-308. · 2.33 Impact Factor
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Alice C N Brown,
Stephane Oddos,
Ian M Dobbie,
Juha-Matti Alakoskela,
Richard M Parton,
Philipp Eissmann,
Mark A A Neil,
Christopher Dunsby, Paul M W French,
Ilan Davis,
Daniel M Davis
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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. · 11.45 Impact Factor
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Clifford B Talbot,
Rakesh Patalay,
Ian Munro,
Sean Warren,
Fulvio Ratto,
Paolo Matteini,
Roberto Pini,
H Georg Breunig,
Karsten König,
Antony C Chu,
Gordon W Stamp,
Mark A A Neil, Paul M W French,
Chris Dunsby
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ABSTRACT: When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo.
Optics Express 07/2011; 19(15):13848-61. · 3.59 Impact Factor
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ABSTRACT: Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.
Biomedical Optics Express 07/2011; 2(7):1907-17. · 2.33 Impact Factor
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ABSTRACT: We present an approach to laser scanning endomicroscopy that requires no moving parts and can be implemented with no distal scanners or optics, permitting extremely compact endoscopic probes to be developed. Our approach utilizes a spatial light modulator to correct for phase variations across a fiber imaging bundle and to encode for arbitrary wavefronts at the distal end of the fiber bundle. Thus, it is possible to realize both focusing and beam scanning at the output of the fiber bundle with no distal components. We present proof of principle results to illustrate three-dimensional scanning of the focal spot and exemplar images of a United States Air Force resolution test chart.
Optics Letters 05/2011; 36(9):1707-9. · 3.40 Impact Factor
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Sunil Kumar,
Dominic Alibhai,
Anca Margineanu,
Romain Laine,
Gordon Kennedy,
James McGinty,
Sean Warren,
Douglas Kelly,
Yuriy Alexandrov,
Ian Munro, [......],
Margaret J Dallman,
Gordon Stamp,
Edward J Murray,
Frank Stuhmeier,
Alessandro Sardini,
Matilda Katan,
Daniel S Elson,
Mark A A Neil,
Chris Dunsby, Paul M W French
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ABSTRACT: A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.
ChemPhysChem 02/2011; 12(3):609-26. · 3.41 Impact Factor
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ABSTRACT: 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.
Biomedical Optics Express 01/2011; 2(5):1340-50. · 2.33 Impact Factor
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Katy L Everett,
Anja Buehler,
Tom D Bunney,
Anca Margineanu,
Rhona W Baxendale,
Petra Vatter,
Michael Retlich,
Claudia Walliser,
Hugh B Manning,
Mark A A Neil,
Christopher Dunsby, Paul M W French,
Peter Gierschik,
Matilda Katan
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ABSTRACT: We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.
Molecular and cellular biology 01/2011; 31(6):1240-51. · 6.06 Impact Factor
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Seden Grippon,
Qiyuan Zhao,
Tom Robinson,
Jacqueline J T Marshall,
Rory J O'Neill,
Hugh Manning,
Gordon Kennedy,
Christopher Dunsby,
Mark Neil,
Stephen E Halford, Paul M W French,
Geoff S Baldwin
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ABSTRACT: Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.
Nucleic Acids Research 11/2010; 39(7):2593-603. · 8.03 Impact Factor
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ABSTRACT: Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organization of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organization of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. The observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling.
Molecular Membrane Biology 08/2010; 27(4-6):178-89. · 2.86 Impact Factor
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ABSTRACT: Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor-bound protein-related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell-APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76-containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.
Science Signaling 01/2010; 3(121):ra36. · 7.50 Impact Factor
