235 results found
Margineanu A, Chan JJ, Kelly DJ, et al., 2016, Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), Scientific Reports, Vol: 6, ISSN: 2045-2322
We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.
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
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.
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.
sikkel MB, kumar S, maioli V, et al., 2015, High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes, Journal of Biophotonics, Vol: 9, Pages: 311-323, ISSN: 1864-0648
blique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure.
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
Gore DM, O'Brart D, Dunsby C, et al., 2015, Transepithelial riboflavin absorption in an ex-vivo rabbit corneal model, Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Publisher: Association for Research in Vision and Ophthalmology, ISSN: 1552-5783
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 IN VIVO 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
Dyer BTB, de Jesus Reis Lagarto J, Sikkel M, et al., 2015, Application of label-free autofluorescence lifetime in vivo to measure changes in myocardial fibrosis and metabolism associated with myocardial infarction and heart failure, EUROPEAN JOURNAL OF HEART FAILURE, Vol: 17, Pages: 367-367, ISSN: 1388-9842
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
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.
Eckersley RJ, Christensen-Jeffries K, Tang MX, et al., 2015, Super-resolution imaging of microbubble contrast agents, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719
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
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
Dyer BT, Lagarto J, French P, et al., 2014, TIME-RESOLVED AUTOFLUORESCENCE SPECTROSCOPY AS LABEL-FREE METHOD TO CHARACTERISE ACUTE CHANGES IN EX VIVO MODELS OF CARDIAC DISEASE, Autumn Meeting of the British-Society-for-Cardiovascular-Research (BSCR) on Cardiovascular Signalling in Health and Disease, Publisher: BMJ PUBLISHING GROUP, ISSN: 1355-6037
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.
Christensen-Jeffries K, Browning RJ, Tang M-X, et al., 2014, In Vivo Acoustic Super-Resolution and Super-Resolved Velocity Mapping Using Microbubbles, IEEE TRANSACTIONS ON MEDICAL IMAGING, Vol: 34, Pages: 433-440, ISSN: 0278-0062
Kelly DJ, Warren SC, Kumar S, et al., 2014, An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays, JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES, Vol: 7, ISSN: 1793-5458
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
Dyer B, Lagarto J, Sikkel M, et al., 2014, THE APPLICATION OF AUTOFLUORESCENCE LIFETIME METROLOGY AS A NOVEL LABEL-FREE TECHNIQUE FOR THE ASSESSMENT OF CARDIAC DISEASE, HEART, Vol: 100, Pages: A104-A104, ISSN: 1355-6037
Gore DM, Margineanu A, French P, et al., 2014, Two-photon fluorescence (TPF) microscopy of corneal riboflavin absorption, Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404
Gore DM, Margineanu A, French P, et al., 2014, Two-Photon Fluorescence Microscopy of Corneal Riboflavin Absorption, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Vol: 55, Pages: 2476-2481, ISSN: 0146-0404
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