Publications
550 results found
Kumar S, Lockward N, Ramel M-C, et 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.
Kim Y, Warren SC, Stone JM, et al., 2016, Adaptive Multiphoton Endomicroscope Incorporating a Polarization-Maintaining Multicore Optical Fibre, IEEE Journal of Selected Topics in Quantum Electronics, Vol: 22, ISSN: 1558-4542
We present a laser scanning multiphoton endomicroscopewith no distal optics or mechanical components that incorporatesa polarization-maintaining (PM) multicore optical fibre todeliver, focus, and scan ultrashort pulsed radiation for two-photonexcited fluorescence imaging. We show theoretically that the use ofa PM multicore fibre in our experimental configuration enhancesthe fluorescence excitation intensity achieved in the focal spot comparedto a non-PM optical fibre with the same geometry and con-firm this by computer simulations based on numerical wavefrontpropagation. In our experimental system, a spatial light modulator(SLM) is utilised to program the phase of the light input to each ofthe cores of the endoscope fibre such that the radiation emergingfrom the distal end of the fibre interferes to provide the focusedscanning excitation beam. We demonstrate that the SLM can enabledynamic phase correction of path-length variations across themulticore optical fibre whilst the fibre is perturbed with an updaterate of 100 Hz.
Maioli V, Gorlitz F, Warren S, et al., 2016, Three-dimensional fluorescence imaging by stage-scanning oblique plane microscopy, Conference on Three-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XXIII, Publisher: SPIE, ISSN: 0277-786X
Andrews N, Ramel M-C, Kumar S, et 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.
Andrews N, Ramel MC, Kumar S, et al., 2016, Fluorescence lifetime optical projection tomography and FRET applied to visualizing apoptosis in live zebrafish larvae
We present the application of FLIM-OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed cleavable FRET biosensor for Caspase-3 as an indicator of gamma radiation induced apoptosis.
Watson TJ, Andrews N, Harry E, et al., 2016, Remote focal scanning and sub-volume optical projection tomography
We present a platform for sub-volume optical projection tomography utilising an electrically tunable lens and tracking technology. Applied to 3D fluorescent bead phantoms and zebrafish embryos, we demonstrate an improvement in resolution and light collection efficiency with respect to conventional optical projection tomography.
sherlock B, Yu F, Stone J, et al., 2016, Tunable fibre-coupled multiphoton microscopy with a negative curvature fibre, Journal of Biophotonics, Vol: 9, Pages: 715-720, ISSN: 1864-0648
Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core andcladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort opticalpulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Itsattenuation was measured to be <0.3 dB.m-1over the range 600-850 nm and the GVD was-180±70 fs2.m-1at 800 nm. Using an average fibre output power of ~20 mW and pulserepetition rate of 80 MHz, the NCF enabled pulses with a duration of <200 fs to be transmittedthrough a length of 1.5 m of fibre over a tuning range of 180 nm without the need for dispersioncompensation. In a 4 m fibre, temporal and spectral pulse widths were maintained to within10% of low power values up to the maximum fibre output power achievable with the lasersystem used of 278 mW at 700 nm, 808 mW at 800 nm and 420 mW at 860 nm. When coupledto a multiphoton microscope, it enabled imaging of ex vivo tissue using excitation wavelengthsfrom 740 nm to 860 nm without any need for adjustments to the set-up.
Kwakwa K, Savell A, Davies T, et al., 2016, easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy, Journal of Biophotonics, Vol: 9, Pages: 948-957, ISSN: 1864-0648
TIRF and STORM microscopy are super-resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low-cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non-TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost.
McGinty J, French P, Frankel P, 2016, Novel 3D imaging platform tracks cancer progression in vivo, Biochemist, Vol: 38, Pages: 12-15, ISSN: 0954-982X
Optical imaging underpins biomedical research in many respects and recent decades have seen spectacular advances, particularly in fluorescence imaging where genetic engineering approaches to labelling have been combined with new light sources, detectors and data analysis techniques to provide capabilities like super-resolution beyond the diffraction limit, exquisite spectroscopic contrast for molecular readouts and high-speed image capture for in vivo and high-throughput applications. However, the main impact of such advanced instrumentation and data analysis has been to provide unprecedented quantitative 2D and 3D information concerning samples compatible with microscopy where volumes of less than 1 mm3 are typically imaged in a single 'acquisition'. The ability to view and measure cellular processes and signalling pathways in live cells has been a significant advance for biomedical research and drug discovery. However, for conventional microscope-based assays and experiments, the samples typically comprise thin layers of cells that are not experiencing the same signals that they would in a 3D tissue context and any findings may not directly translate to live organisms. It is desirable to study disease processes in live intact organisms that can provide appropriate physiological complexity. For cancer studies, recent research from our group shows that optical tomography can be used to directly monitor in vivo changes in tumour growth and vascular development in a zebrafish cancer model over time. This technique not only improves the value of the collected data, but if used on a wider scale should result in a reduction in the number of animals used in biomedical research.
Kumar S, Lockwood N, Ramel MC, et al., 2016, In vivo multiplexed OPT and FLIM OPT of an adult zebrafish cancer disease model
We report angular multiplexed OPT and FLIM OPT applied to in vivo imaging of cancer and FRET biosensors in adult zebrafish. Multiple-spectral 3-D datasets of entire adult zebrafish can be acquired in 3 minutes.
Perdios L, Bunney TD, Warren SC, et al., 2016, Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1., Advances in Biological Regulation, Vol: 60, Pages: 6-13, ISSN: 2212-4934
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.
Gore DM, O'Brart DP, French P, et al., 2015, A Comparison of Different Corneal Iontophoresis Protocols for Promoting Transepithelial Riboflavin Penetration., Investigative Ophthalmology & Visual Science, Vol: 56, Pages: 7908-7914, ISSN: 1552-5783
PURPOSE: To measure corneal riboflavin penetration using different transepithelial iontophoresis protocols. METHODS: Freshly enucleated rabbit eyes were divided into nine treatment groups of 4 eyes. One group, in which 0.1% wt/vol riboflavin was applied for 30 minutes without iontophoresis after corneal epithelial debridement, acted as a control. The remaining groups were treated with an intact epithelium using different riboflavin formulations and varying iontophoresis current, soak, and rinse times. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35 μm thick were then imaged immediately by two-photon fluorescence microscopy, using image processing software to quantify stromal riboflavin concentration at different corneal depths. RESULTS: In the epithelium-on iontophoresis treatment groups, greater stromal riboflavin penetration was achieved with higher-concentration riboflavin solutions, greater iontophoresis dosage, and longer solution contact times. A protocol utilizing 0.25% wt/vol riboflavin with benzalkonium chloride (BAC) 0.01% and two cycles of applied current and subsequent soaking (1 mA 5 minutes, soak 5 minutes; 0.5 mA 5 minutes, soak 5 minutes) achieved similar stromal riboflavin penetration to epithelium-off controls. The best-performing non-BAC-containing protocol produced stromal riboflavin penetration approximately 60% that of epithelium-off controls. Riboflavin solutions containing saline resulted in minimal stromal penetration. Riboflavin loading within the epithelium was equivalent to or higher than that in the subjacent stroma, despite rinsing the ocular surface with balanced salt solution. CONCLUSIONS: Modified iontophoresis protocols can significantly improve transepithelial riboflavin penetration in experimental corneal collagen cross-linking.
Roper JC, Yerolatsitis S, Birks TA, et al., 2015, Minimizing Group Index Variations in a Multicore Endoscope Fiber, IEEE Photonics Technology Letters, Vol: 27, Pages: 2359-2362, ISSN: 1941-0174
We describe a multicore fiber for nonlinear endoscopy where an ultrashort pulse is divided up and delivered through multiple cores to reduce the peak power. The variation in group index between cores of the fiber is minimized to allow simultaneously launched sub-pulses to arrive at the distal end of the fiber synchronously. Minimization of group index variation between cores is achieved at a V parameter of 3 owing to a turning point in the relationship between the group index and V parameter. For synchronized arrival times, single-mode propagation is important. By tapering a short length of the fiber at the launch end, the V value is locally brought below 2.405 allowing a pure fundamental mode to be launched into each core.
Correia T, Lockwood N, Kumar S, et 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.
Dyer BT, Elder JM, Lagarto J, et al., 2015, Application of label-free autofluorescence lifetime in vivo to measure changes in myocardial fibrosis and metabolism in a doxorubicin cardiomyopathy heart failure model, Congress of the European-Society-of-Cardiology (ESC), Publisher: OXFORD UNIV PRESS, Pages: 151-151, ISSN: 0195-668X
Gore DM, O'Brart D, French P, et al., 2015, Transepithelial Riboflavin Absorption in an Ex Vivo Rabbit Corneal Model, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Vol: 56, Pages: 5006-5011, ISSN: 0146-0404
Warren SC, Margineanu A, Katan M, et al., 2015, Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells., International Journal of Molecular Sciences, Vol: 16, Pages: 14695-14716, ISSN: 1661-6596
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.
Talbot CB, Lagarto J, Warren S, et al., 2015, Correction Approach for Delta Function Convolution Model Fitting of Fluorescence Decay Data in the Case of a Monoexponential Reference Fluorophore, Journal of Fluorescence, Vol: 25, Pages: 1169-1182, ISSN: 1573-4994
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.
Dyer BT, Elder JM, Lagarto J, et al., 2015, LABEL-FREE AUTOFLUORESCENCE LIFETIME TO ASSESS CHANGES IN MYOCARDIAL FIBROSIS AND METABOLISM <i>IN VIVO</i> IN A DOXORUBICIN CARDIOMYOPATHY HEART FAILURE MODEL, British-Cardiac-Society (BCS) Annual Conference on Hearts and Genes, Publisher: BMJ PUBLISHING GROUP, Pages: A94-A94, ISSN: 1355-6037
Sherlock B, Warren S, Stone J, et al., 2015, Fibre-coupled multiphoton microscope with adaptive motion compensation, BIOMEDICAL OPTICS EXPRESS, Vol: 6, Pages: 1876-1884, ISSN: 2156-7085
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- Citations: 8
McGinty J, Chen L, Kumar S, et al., 2015, Techniques to improve the spatial and temporal resolution in optical projection tomography: Remote focal scanning and time-lapse cell tracking
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.
Chen L, Alexandrov Y, Kumar S, et al., 2015, Mesoscopic <i>in vivo</i> 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography, BIOMEDICAL OPTICS EXPRESS, Vol: 6, Pages: 1253-1261, ISSN: 2156-7085
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- Citations: 7
Mitchell RK, Mondragon A, Chen L, et al., 2015, Selective disruption of <i>Tcf7l2</i> in the pancreatic β cell impairs secretory function and lowers β cell mass, HUMAN MOLECULAR GENETICS, Vol: 24, Pages: 1390-1399, ISSN: 0964-6906
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- Citations: 75
Sayers S, Kantor C, Pullen TJ, et al., 2015, Preserved insulin secretion despite impaired glucose signalling after pancreatic beta cell selective deletion of the tumour suppressor LKB1, Publisher: WILEY-BLACKWELL, Pages: 13-13, ISSN: 0742-3071
Gore DM, French P, O'Brart D, et al., 2015, Two-Photon Fluorescence Microscopy of Corneal Riboflavin Absorption Through an Intact Epithelium, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Vol: 56, Pages: 1191-1192, ISSN: 0146-0404
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- Citations: 6
Lagarto J, Dyer BT, Talbot C, et al., 2015, Application of time-resolved autofluorescence to label-free in vivo optical mapping of changes in tissue matrix and metabolism associated with myocardial infarction and heart failure, Biomedical Optics Express, Vol: 6, Pages: 324-346, ISSN: 2156-7085
We investigate the potential of an instrument combining timeresolvedspectrofluorometry and diffuse reflectance spectroscopy tomeasure structural and metabolic changes in cardiac tissue in vivo in a 16week post-myocardial infarction heart failure model in rats. In the scarregion, we observed changes in the fluorescence signal that can beexplained by increased collagen content, which is in good agreement withhistology. In areas remote from the scar tissue, we measured changes in thefluorescence signal (p < 0.001) that cannot be explained by differences incollagen content and we attribute this to altered metabolism within themyocardium. A linear discriminant analysis algorithm was applied to themeasurements to predict the tissue disease state. When we combine allmeasurements, our results reveal high diagnostic accuracy in the infarctedarea (100%) and border zone (94.44%) as well as in remote regions fromthe scar (> 77%). Overall, our results demonstrate the potential of ourinstrument to characterize structural and metabolic changes in a failing heartin vivo without using exogenous labels.
Tatla T, Sparks H, Charn T, et al., 2015, Development of endoscopic fluorescence lifetime imaging microscopy (FLIM) for head and neck squamous cell cancer (HNSCC) screening: sub-site ex vivo data analysis, Head and Neck Optical Diagnostic Society Annual Meeting
Nolte DD, Jeong K, Turek J, et al., 2015, Holographic optical coherence imaging, Optical Coherence Tomography: Technology and Applications, Second Edition, Pages: 941-964, ISBN: 9783319064185
This chapter gives an overview of the principles of holographic OCI. It begins with a description of off-axis holography as spatial heterodyne detection and continues with the origin and role of speckle in multichannel illumination of tissue. Image-domain holography (IDH) and Fourier–domain holography (FDH) are described. Holography in the Fourier domain has the capability for phase–contrast imaging that can acquire small sub–wavelength displacements despite long coherence length. The trade–offs between photorefractive and digital holography are discussed. The chief biological target is multicellular spheroids, specifically rat osteogenic sarcomas that are grown in vitro. After describing the physiological and optical properties of these spheroids, results from holographic OCI are presented using both photorefractive and digital holography.
McGinty J, Chen L, Kumar S, et al., 2015, Techniques to improve the spatial and temporal resolution in optical projection tomography: Remote focal scanning and time-lapse cell tracking
© OSA 2015. 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.
Kelly DJ, Warren SC, Alibhai D, et al., 2015, Automated multiwell fluorescence lifetime imaging for Forster resonance energy transfer assays and high content analysis, ANALYTICAL METHODS, Vol: 7, Pages: 4071-4089, ISSN: 1759-9660
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- Citations: 10
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