Publications
300 results found
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.
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.
Görlitz 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.
Casey D, Wylie D, Neil M, 2017, Optical techniques and microtools for subcellular delivery and sampling, LIGHT ROBOTICS STRUCTURE-MEDIATED NANOBIOPHOTONICS, Publisher: ELSEVIER SCIENCE BV, Pages: 287-311, ISBN: 978-0-7020-7096-9
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.
Warren SC, Kim Y, Stone JM, et al., 2016, Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection, Optics Express, Vol: 24, Pages: 21474-21484, ISSN: 1094-4087
This paper demonstrates multiphoton excited fluorescenceimaging through a polarisation maintaining multicore fiber (PM-MCF)while the fiber is dynamically deformed using all-proximal detection.Single-shot proximal measurement of the relative optical path lengths of allthe cores of the PM-MCF in double pass is achieved using a Mach-Zehnderinterferometer read out by a scientific CMOS camera operating at 416 Hz.A non-linear least squares fitting procedure is then employed to determinethe deformation-induced lateral shift of the excitation spot at the distal tip ofthe PM-MCF. An experimental validation of this approach is presented thatcompares the proximally measured deformation-induced lateral shift infocal spot position to an independent distally measured ground truth. Theproximal measurement of deformation-induced shift in focal spot position isapplied to correct for deformation-induced shifts in focal spot positionduring raster-scanning multiphoton excited fluorescence imaging.
Friddin MS, Bolognesi G, Elani Y, et al., 2016, The optical assembly of bilayer networks from cell-sized droplets for synthetic biology, Systems and Synthetic Biology
Friddin MS, Bolognesi G, Elani Y, et al., 2016, Optical tweezers to assemble 2D and 3D droplet interface bilayer networks from cell-sized droplets, EMBL Microfluidics
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.
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.
Friddin MS, Bolognesi G, Elani Y, et al., 2016, Light-driven drag and drop assembly of micron-scale bilayer networks for synthetic biology, Pages: 545-546
We have developed a new method to assemble single- or multi-layered networks of droplet interface bilayers (DIBs) from cell-sized droplets using a single beam optical trap (optical tweezers). The novelty of our approach is the ability to directly trap the microdroplets with the laser and manipulate them in 3D to construct DIB networks of user-defined architectures. Our method does not require a complex optical setup, is versatile, contactless, benefits from both high spatial and temporal resolution, and could set a new paradigm for the assembly of smart, synthetic biosystems.
Willison KR, Salehi-Reyhani A, Burgin E, et al., 2015, Absolute quantification of protein copy number in single cells using single molecule microarrays, EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, Vol: 44, Pages: S179-S179, ISSN: 0175-7571
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.
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
Casey D, Wylie D, Gallo J, et al., 2015, A novel, all-optical tool for controllable and non-destructive poration of cells with single-micron resolution, Bio-Optics: Design and Application 2015, Publisher: Optical Society of America
We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience.
Koleva MV, Rothery S, Spitaler M, et al., 2015, Sonic hedgehog multimerization: A self-organizing event driven by post-translational modifications?, MOLECULAR MEMBRANE BIOLOGY, Vol: 32, Pages: 65-74, ISSN: 0968-7688
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- Citations: 8
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
Bolognesi G, Hargreaves A, Ward AD, et al., 2015, Microfluidic generation and optical manipulation of ultra-low interfacial tension droplets, Conference on Integrated Photonics - Materials, Devices, and Applications III, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
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- Citations: 1
Robinson T, Valluri P, Kennedy G, et al., 2014, Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy, Analytical Chemistry, Vol: 86, Pages: 10732-10740, ISSN: 0003-2700
Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.
Schrems A, Phillips J, Casey D, et al., 2014, The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells (vol 139, pg 3296, 2014), ANALYST, Vol: 139, Pages: 4382-4382, ISSN: 0003-2654
Schrems A, Phillips J, Casey D, et al., 2014, Erratum: The grab-and-drop protocol: A novel strategy for membrane protein isolation and reconstitution from single cells (Analyst (2014) DOI: 10.1039/C4AN00059E), Analyst, Vol: 139, ISSN: 0003-2654
Sonnefraud Y, Sinclair HG, Sivan Y, et al., 2014, Experimental Proof of Concept of Nanoparticle-Assisted STED, NANO LETTERS, Vol: 14, Pages: 4449-4453, ISSN: 1530-6984
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- Citations: 26
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