284 results found
Lightley J, Gorlitz F, Kumar S, et al., 2021, Robust deep learning optical autofocus system applied to automated multiwell plate single molecule localization microscopy, JOURNAL OF MICROSCOPY, ISSN: 0022-2720
Gong H, Guo W, Neil MAA, 2021, GPU-accelerated real-time reconstruction in Python of three-dimensional datasets from structured illumination microscopy with hexagonal patterns, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY A-MATHEMATICAL PHYSICAL AND ENGINEERING SCIENCES, Vol: 379, ISSN: 1364-503X
Boland MA, Cohen EAK, Flaxman SR, et al., 2021, Improving axial resolution in Structured Illumination Microscopy using deep learning, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY A-MATHEMATICAL PHYSICAL AND ENGINEERING SCIENCES, Vol: 379, ISSN: 1364-503X
Garcia E, Lightley J, Kumar S, et al., 2021, Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease, JOURNAL OF PATHOLOGY CLINICAL RESEARCH, Vol: 7, Pages: 438-445
Jones DC, Alexandrov Y, Curry N, et al., 2021, Multidimensional spectroscopy and imaging of defects in synthetic diamond: excitation-emission-lifetime luminescence measurements with multiexponential fitting and phasor analysis, Journal of Physics D: Applied Physics, Vol: 54, Pages: 1-13, ISSN: 0022-3727
We report the application of phasor analysis and nonlinear iterative fitting to complex spatial and spectroscopic luminescence decay data obtained from multidimensional microscopy of a CVD diamond grown on a HPHT substrate. This spectral and lifetime-resolved analysis enabled spatial mapping of variations in concentrations of nitrogen vacancy (NV) defects in both charge states and the quenching of NV− defects, as well as the identification of SiV− luminescence. These imaging and spectroscopic modalities may be important for reliable fabrication of quantum devices based on such defects in diamond, which will require well-defined and characterised quantum electronic properties.
Quicke P, Howe CL, Song P, et al., 2020, Subcellular resolution 3D light field imaging with genetically encoded voltage indicators, Neurophotonics, Vol: 7, ISSN: 2329-4248
Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM’s volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity.Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions.Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM.Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM.Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM’s potential for studying dendritic integration and action potential propagation in three spatial dimensions.
Quicke P, Howe CL, Song P, et al., 2020, Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators, Neurophotonics, Vol: 7, ISSN: 2329-4248
Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM's volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity. Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions. Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM. Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM. Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM's potential for studying dendritic integration and action potential propagation in three spatial dimensions.
Gorlitz F, Wysoczanski R, Kumar S, et al., 2020, Towards easier, faster super-resolved microscopy, Conference on Single Molecule Spectroscopy and Superresolution Imaging XIII, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Jones DC, Kumar S, Lanigan PMP, et al., 2019, Multidimensional luminescence microscope for imaging defect colour centres in diamond, Methods and Applications in Fluorescence, Vol: 8, ISSN: 2050-6120
We report a multidimensional luminescence microscope providing hyperspectral imaging and time-resolved (luminescence lifetime) imaging for the study of luminescent diamond defects. The instrument includes crossed-polariser white light transmission microscopy to reveal any birefringence that would indicate strain in the diamond lattice. We demonstrate the application of this new instrument to defects in natural and synthetic diamonds including N3, nitrogen and silicon vacancies. Hyperspectral imaging provides contrast that is not apparent in conventional intensity images and the luminescence lifetime provides further contrast.
Quicke P, Song C, McKimm EJ, et al., 2019, Corrigendum: Single-neuron level one-photon voltage imaging with sparsely targeted genetically encoded voltage indicators, Frontiers in Cellular Neuroscience, Vol: 13, ISSN: 1662-5102
Voltage imaging of many neurons simultaneously at single-cell resolution is hampered bythe difficulty of detecting small voltage signals from overlapping neuronal processes inneural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaginghave shown single-cell resolution optical voltage recordings in intact tissue throughimaging naturally sparse cell classes, sparse viral expression, soma restricted expression,advanced optical systems, or a combination of these. Widespread sparse and strongtransgenic GEVI expression would enable straightforward optical access to a denselyoccurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recentlydescribed sparse transgenic expression strategy can enable single-cell resolution voltageimaging of cortical pyramidal cells in intact brain tissue without restricting expression tothe soma. We also quantify the functional crosstalk in brain tissue and discuss optimalimaging rates to inform future GEVI experimental design.
Soor N, Quicke P, Howe C, et al., 2019, All-optical crosstalk-free manipulation and readout of Chronos-expressing neurons, Journal of Physics D: Applied Physics, Vol: 52, Pages: 1-10, ISSN: 0022-3727
All optical neurophysiology allows manipulation and readout of neural network activity with single-cell spatial resolution and millisecond temporal resolution. Neurons can be made to express proteins that actuate transmembrane currents upon light absorption, enabling optical control of membrane potential and action potential signalling. In addition, neurons can be genetically or synthetically labelled with fluorescent reporters of changes in intracellular calcium concentration or membrane potential. Thus, to optically manipulate and readout neural activity in parallel, two spectra are involved: the action spectrum of the actuator, and the absorption spectrum of the fluorescent reporter. Due to overlap in these spectra, previous all-optical neurophysiology paradigms have been hindered by spurious activation of neuronal activity caused by the readout light. Here, we pair the blue-green absorbing optogenetic actuator, Chronos, with a deep red-emitting fluorescent calcium reporter CaSiR-1. We show that cultured Chinese hamster ovary cells transfected with Chronos do not exhibit transmembrane currents when illuminated with wavelengths and intensities suitable for exciting one-photon CaSiR-1 fluorescence. We then demonstrate crosstalk-free, high signal-to-noise ratio CaSiR-1 red fluorescence imaging at 100 frames s−1 of Chronos-mediated calcium transients evoked in neurons with blue light pulses at rates up to 20 Hz. These results indicate that the spectral separation between red light excited fluorophores, excited efficiently at or above 640 nm, with blue-green absorbing opsins such as Chronos, is sufficient to avoid spurious opsin actuation by the imaging wavelengths and therefore enable crosstalk-free all-optical neuronal manipulation and readout.
Quicke P, Song C, McKimm EJ, et al., 2019, Single-neuron level one-photon voltage imaging with sparsely targeted genetically encoded voltage indicators, Frontiers in Cellular Neuroscience, Vol: 13, ISSN: 1662-5102
Voltage imaging of many neurons simultaneously at single-cell resolution is hampered by the difficulty of detecting small voltage signals from overlapping neuronal processes in neural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaging have shown single-cell resolution optical voltage recordings in intact tissue through imaging naturally sparse cell classes, sparse viral expression, soma restricted expression, advanced optical systems, or a combination of these. Widespread sparse and strong transgenic GEVI expression would enable straightforward optical access to a densely occurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recently described sparse transgenic expression strategy can enable single-cell resolution voltage imaging of cortical pyramidal cells in intact brain tissue without restricting expression to the soma. We also quantify the functional crosstalk in brain tissue and discuss optimal imaging rates to inform future GEVI experimental design.
Munro I, Garcia EAC, Yan M, et al., 2019, Accelerating single molecule localisation microscopy through parallel processing on a high-performance computing cluster, Journal of Microscopy, Vol: 273, Pages: 148-160, ISSN: 1365-2818
Super‐resolved microscopy techniques have revolutionized the ability to study biological structures below the diffraction limit. Single molecule localization microscopy (SMLM) techniques are widely used because they are relatively straightforward to implement and can be realized at relatively low cost, e.g. compared to laser scanning microscopy techniques. However, while the data analysis can be readily undertaken using open source or other software tools, large SMLM data volumes and the complexity of the algorithms used often lead to long image data processing times that can hinder the iterative optimization of experiments. There is increasing interest in high throughput SMLM, but its further development and application is inhibited by the data processing challenges. We present here a widely applicable approach to accelerating SMLM data processing via a parallelized implementation of ThunderSTORM on a high‐performance computing (HPC) cluster and quantify the speed advantage for a four‐node cluster (with 24 cores and 128 GB RAM per node) compared to a high specification (28 cores, 128 GB RAM, SSD‐enabled) desktop workstation. This data processing speed can be readily scaled by accessing more HPC resources. Our approach is not specific to ThunderSTORM and can be adapted for a wide range of SMLM software.
Quicke P, Howe CL, Song P, et al., 2019, Calculation of high numerical aperture lightfield microscope point spread functions
3D deconvolution of lightfield images enables high resolution reconstruction of sample volumes. Previous point spread function calculations assume low to moderate NA objectives. Here we present a simple vectorial calculation valid for high NA objectives.
Soltan A, Maaskant P, Armstrong N, et al., 2019, Wearable Glasses for Retinal Pigmentiosa Based on Optogenetics, IEEE International Symposium on Circuits and Systems (IEEE ISCAS), Publisher: IEEE, ISSN: 0271-4302
Gorlitz F, Lightley J, Kumar S, et al., 2019, Automated multiwell plate STORM: towards open source super-resolved high content analysis, Conference on Advances in Microscopic Imaging II, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Gorlitz F, Guldbrand S, Runcorn T, et al., 2018, easySLM-STED: stimulated emission depletion microscopy with aberration correction, extended field of view and multiple beam scanning, Journal of Biophotonics, Vol: 11, ISSN: 1864-063X
We demonstrate a simplified set‐up for STED microscopy with a straightforward alignment procedure that uses a single spatial light modulator (SLM) with collinear incident excitation and depletion beams to provide phase modulation of the beam profiles and correction of optical aberrations. We show that this approach can be used to extend the field of view for STED microscopy by correcting chromatic aberration that otherwise leads to walk‐off between the focused excitation and depletion beams. We further show how this arrangement can be adapted to increase the imaging speed through multibeam excitation and depletion. Fine adjustments to the alignment can be accomplished using the SLM only, conferring the potential for automation.
Soltan A, Barrett JM, Maaskant P, et al., 2018, A head mounted device stimulator for optogenetic retinal prosthesis, Journal of Neural Engineering, Vol: 15, ISSN: 1741-2552
Objective. Our main objective is to demonstrate that compact high radiance gallium nitride displays can be used with conventional virtual reality optics to stimulate an optogenetic retina. Hence, we aim to introduce a non-invasive approach to restore vision for people with conditions such as retinitis pigmentosa where there is a remaining viable communication link between the retina and the visual cortex. Approach. We design and implement the headset using a high-density µLED matrix, Raspberry Pi, microcontroller from NXP and virtual reality lens. Then, a test platform is developed to evaluate the performance of the headset and the optical system. Furthermore, image simplification algorithms are used to simplify the scene to be sent to the retina. Moreover, in vivo evaluation of the genetically modified retina response at different light intensity is discussed to prove the reliability of the proposed system. Main results. We demonstrate that in keeping with regulatory guidance, the headset displays need to limit their luminance to 90 kcd m−2. We demonstrate an optical system with 5.75% efficiency which allows for 0.16 mW mm−2 irradiance on the retina within the regulatory guidance, but which is capable of an average peak irradiance of 1.35 mW mm−2. As this is lower than the commonly accepted threshold for channelrhodopsin-2, we demonstrate efficacy through an optical model of an eye onto a biological retina. Significance. We demonstrate a fully functional 8100-pixel headset system including software/hardware which can operate on a standard consumer battery for periods exceeding a 24 h recharge cycle. The headset is capable of delivering enough light to stimulate the genetically modified retina cells and also keeping the amount of light below the regulation threshold for safety.
Quicke P, Reynolds S, Neil M, et al., 2018, High speed functional imaging with source localized multifocal two-photon microscopy, Biomedical Optics Express, Vol: 9, Pages: 3678-3693, ISSN: 2156-7085
Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.
Kim Y, Warren S, Favero F, et al., 2018, Semi-random multicore fibre design for adaptive multiphoton endoscopy, Optics Express, Vol: 26, Pages: 3661-3673, ISSN: 1094-4087
This paper reports the development, modelling and application of a semi-random multicore fibre (MCF) design for adaptive multiphoton endoscopy. The MCF was constructed from 55 sub-units, each comprising 7 single mode cores, in a hexagonally close-packed lattice where each sub-unit had a random angular orientation. The resulting fibre had 385 single mode cores and was double-clad for proximal detection of multiphoton excited fluorescence. The random orientation of each sub-unit in the fibre reduces the symmetry of the positions of the cores in the MCF, reducing the intensity of higher diffracted orders away from the central focal spot formed at the distal tip of the fibre and increasing the maximum size of object that can be imaged. The performance of the MCF was demonstrated by imaging fluorescently labelled beads with both distal and proximal fluorescence detection and pollen grains with distal fluorescence detection. We estimate that the number of independent resolution elements in the final image – measured as the half-maximum area of the two-photon point spread function divided by the area imaged – to be ~3200.
Quicke P, Neil M, Knopfel T, et al., 2017, Source-Localized Multifocal Two-Photon Microscopy for High-Speed Functional Imaging, 71st Annual Meeting of the Society-of-General-Physiologists (SGP) on Optical Revolution in Physiology - From Membrane to Brain, Publisher: ROCKEFELLER UNIV PRESS, Pages: 13A-14A, ISSN: 0022-1295
Sherlock B, Warren SC, Alexandrov Y, et al., 2017, In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation, Journal of Biophotonics, Vol: 11, ISSN: 1864-063X
This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to ~65 μm s-1. Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.
Gorlitz F, Corcoran DS, Garcia Castano EA, et al., 2017, Mapping molecular function to biological nanostructure: combining structured illumination microscopy with fluorescence lifetime imaging (SIM+FLIM), Photonics, Vol: 4, ISSN: 2304-6732
We present a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. We illustrate this SIM + FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle.
Casey D, Wylie D, Neil M, 2017, Optical techniques and microtools for subcellular delivery and sampling, Light Robotics-Structure-Mediated Nanobiophotonics, Pages: 287-311, ISBN: 9780702070969
The vast majority of biochemical and histological assays are conducted upon ensembles of tens or hundreds or thousands of cells. However, there is a robust and increasing volume of evidence that small subpopulations of cells play significant roles in the development of many disease states, and particularly in their evolution toward treatment resistance. As such, a novel suite of optical tools has been and is being developed, offering functionality analogous to traditional techniques but furnishing them with single- or even subcellular resolution. These are designed to probe the operations of life at its most fundamental level, providing insights into the complex, crowded, and seemingly chaotic processes underpinning cellular processes and their malfunctions.In this chapter, the development of these tools will be explored from their origins to their current state of the art, alongside their applications in both proof-of-concept and applied clinical roles. The focus will be upon tools and techniques designed and developed to interact directly with the cell membrane, taking in optical lysis, poration, and fusion techniques alongside surface chemistry and thin-film coatings capable of causing structural effects within a target cell's plasma membrane. Notable successes and problems will be critically analyzed, and the prospects for the future will be discussed with reference to emerging technologies and promising research avenues in the field.
Soltan A, McGovern B, Drakakis E, et al., 2017, High density, high radiance mu LED matrix for optogenetic retinal Prostheses and planar neural stimulation, IEEE Transactions on Biomedical Circuits and Systems, Vol: 11, Pages: 347-359, ISSN: 1932-4545
Optical neuron stimulation arrays are important for both in-vitro biology and retinal prosthetic biomedical applications. Hence, in this work, we present an 8100 pixel high radiance photonic stimulator. The chip module vertically combines custom made gallium nitride μLEDs with a CMOS application specific integrated circuit. This is designed with active pixels to ensure random access and to allow continuous illumination of all required pixels. The μLEDs have been assembled on the chip using a solder ball flip-chip bonding technique which has allowed for reliable and repeatable manufacture. We have evaluated the performance of the matrix by measuring the different factors including the static, dynamic power consumption, the illumination, and the current consumption by each LED. We show that the power consumption is within a range suitable for portable use. Finally, the thermal behavior of the matrix is monitored and the matrix proved to be thermally stable.
Sparks H, Gorlitz F, Kelly D, et al., 2017, Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging, Review of Scientific Instruments, Vol: 88, ISSN: 1089-7623
We report the characterisation of gated optical image intensifiers for fluorescence lifetime imaging (FLIM), evaluating the performance of several different prototypes that culminate in a new design that provides improved spatial resolution conferred by the addition of a magnetic field to reduce the lateral spread of photoelectrons on their path between the photocathode and microchannel plate, and higher signal to noise ratio conferred by longer time gates. We also present a methodology to compare thesesystems and their capabilities, including the quantitative readouts of Förster resonant energy transfer (FRET).
French PMW, Görlitz F, Kelly D, et al., 2017, Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy, Jove-Journal of Visualized Experiments, Vol: 119, ISSN: 1940-087X
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
Go¨rlitz F, Corcoran DS, Sparks H, et al., 2017, Mapping molecular function to biological nanostructure: Combining structured illumination microscopy with fluorescence lifetime imaging
We report the enhancement of spatial resolution and sensitivity of wide-field time-gated imaging and the combination with SIM to map molecular function using FRET to biological nanostructure below the conventional diffraction limit.
Cortés E, Huidobro PA, Sinclair HG, et al., 2016, Plasmonic nanoprobes for stimulated emission depletion nanoscopy, ACS Nano, Vol: 10, Pages: 10454-10461, ISSN: 1936-0851
Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm. These particles provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power. The nanoparticle-assisted STED probes reported here represent a ∼2 × 103 reduction in probe volume compared to previously used nanoparticles. Finally, we demonstrate their application toward plasmon-assisted STED cellular imaging at low-depletion powers, and we also discuss their current limitations.
Friddin MS, Bolognesi G, Elani Y, et al., 2016, Optically assembled droplet interface bilayer (OptiDIB) networks from cell-sized microdroplets, Soft Matter, Vol: 12, Pages: 7731-7734, ISSN: 1744-6848
We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems.
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