Imperial College London

Dr Chris Dunsby

Faculty of Natural SciencesDepartment of Physics

Professor of Biomedical Optics
 
 
 
//

Contact

 

+44 (0)20 7594 7755christopher.dunsby Website

 
 
//

Location

 

622Blackett LaboratorySouth Kensington Campus

//

Summary

 

Publications

Publication Type
Year
to

235 results found

Sparks H, Kondo H, Hooper S, Munro I, Kennedy G, Dunsby C, French P, Sahai Eet al., 2018, Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy, Nature Communications, Vol: 9, ISSN: 2041-1723

We present an approach to quantify drug-target engagement using in vivo fluorescence endomicroscopy, validated with in vitro measurements. Doxorubicin binding to chromatin changes the fluorescence lifetime of histone-GFP fusions that we measure in vivo at single-cell resolution using a confocal laparo/endomicroscope. We measure both intra- and inter-tumor heterogeneity in doxorubicin chromatin engagement in a model of peritoneal metastasis of ovarian cancer, revealing striking variation in the efficacy of doxorubicin-chromatin binding depending on intra-peritoneal or intravenous delivery. Further, we observe significant variations in doxorubicin-chromatin binding between different metastases in the same mouse and between different regions of the same metastasis. The quantitative nature of fluorescence lifetime imaging enables direct comparison of drug-target engagement for different drug delivery routes and between in vitro and in vivo experiments. This uncovers different rates of cell killing for the same level of doxorubicin binding in vitro and in vivo.

Journal article

Zhang G, Harput S, Lin S, Christensen-Jeffries K, Leow CH, Brown J, Dunsby C, Eckersley R, Tang Met al., 2018, Acoustic wave sparsely-activated localization microscopy (AWSALM): super-resolution ultrasound imaging using acoustic activation and deactivation of nanodroplets, Applied Physics Letters, Vol: 113, ISSN: 1077-3118

Photo-activated localization microscopy (PALM) has revolutionized the field of fluorescence microscopy by breaking the diffraction limit in spatial resolution. In this study, “acoustic wave sparsely activated localization microscopy (AWSALM),” an acoustic counterpart of PALM, is developed to super-resolve structures which cannot be resolved by conventional B-mode imaging. AWSALM utilizes acoustic waves to sparsely and stochastically activate decafluorobutane nanodroplets by acoustic vaporization and to simultaneously deactivate the existing vaporized nanodroplets via acoustic destruction. In this method, activation, imaging, and deactivation are all performed using acoustic waves. Experimental results show that sub-wavelength micro-structures not resolvable by standard B-mode ultrasound images can be separated by AWSALM. This technique is flow independent and does not require a low concentration of contrast agents, as is required by current ultrasound super resolution techniques. Acoustic activation and deactivation can be controlled by adjusting the acoustic pressure, which remains well within the FDA approved safety range. In conclusion, this study shows the promise of a flow and contrast agent concentration independent super-resolution ultrasound technique which has potential to be faster and go beyond vascular imaging.

Journal article

Alexandrov Y, Nikolic DS, Dunsby C, French PMWet al., 2018, Quantitative time domain analysis of lifetime-based FRET measurements with fluorescent proteins: static random isotropic fluorophore orientation distributions, Journal of Biophotonics, Vol: 11, ISSN: 1864-063X

Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime-based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.

Journal article

Harput S, Christensen-Jeffries K, Brown J, Yuanwei L, Williams KJ, Davies AH, Eckersley R, Dunsby CW, Tang Met al., 2018, Two-stage motion correction for super-resolution ultrasound imaging in human lower limb, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 65, Pages: 803-814, ISSN: 0885-3010

The structure of microvasculature cannot be resolved using conventional ultrasound imaging due to the fundamental diffraction limit at clinical ultrasound frequencies. It is possible to overcome this resolution limitation by localizing individual microbubbles through multiple frames and forming a super-resolved image, which usually requires seconds to minutes of acquisition. Over this time interval, motion is inevitable and tissue movement is typically a combination of large and small scale tissue translation and deformation. Therefore, super-resolution imaging is prone to motion artefacts as other imaging modalities based on multiple acquisitions are. This study investigates the feasibility of a two-stage motion estimation method, which is a combination of affine and non-rigid estimation, for super-resolution ultrasound imaging. Firstly, the motion correction accuracy of the proposed method is evaluated using simulations with increasing complexity of motion. A mean absolute error of 12.2 μm was achieved in simulations for the worst case scenario. The motion correction algorithm was then applied to a clinical dataset to demonstrate its potential to enable in vivo super-resolution ultrasound imaging in the presence of patient motion. The size of the identified microvessels from the clinical super-resolution images were measured to assess the feasibility of the two-stage motion correction method, which reduced the width of the motion blurred microvessels approximately 1.5-fold.

Journal article

Kim Y, Warren S, Favero F, Stone J, Clegg J, Neil M, Paterson C, Knight J, French P, Dunsby CWet 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.

Journal article

Harput S, Christensen-Jeffries K, Brown J, Eckersley RJ, Dunsby C, Tang M-Xet al., 2017, Ultrasound Super-Resolution with Microbubble Contrast Agents, 16th IEEE SENSORS CONFERENCE, Publisher: IEEE, Pages: 1104-1106, ISSN: 1930-0395

Ultrasound super-resolution imaging can be achieved by localizing spatially isolated microbubble contrast agents over multiple imaging frames. In vivo images with resolutions of ~10-20 microns in deep tissue have been demonstrated. The technique has the potential to revolutionize the way micro-circulation can be visualized and quantified, and has implications in a wide range of clinical applications including cancer, diabetes and beyond. In this paper we describe the principle of the technique with in vivo results demonstrating the superior resolution achieved compared with existing ultrasound imaging. We also discuss the challenges and opportunities in the area of 3D imaging including, imaging speed, tissue motion and microbubble localization errors.

Conference paper

Harput S, Christensen-Jeffries K, Brown J, Eckersley RJ, Dunsby C, Tang MXet al., 2017, Localisation of multiple non-isolated microbubbles with frequency decomposition in super-resolution imaging, IEEE International Ultrasonics Symposium, IUS, Publisher: IEEE, ISSN: 1948-5719

Sub-diffraction imaging, also known as ultrasound localization microscopy, is a novel method that can overcome the fundamental diffraction limit by localizing spatially isolated microbubbles. This method requires the use of a low concentration of microbubbles to ensure that they are spatially isolated. For in vivo microvascular imaging, especially for cancer tissue with high microvascular density, spatial isolation cannot be always achieved, since vessels are close to each other and the speed of flow is slow. This study proposes a frequency decomposition method that uses the polydisperse nature of commercial contrast agents to separate spatially non-isolated microbubbles with different acoustic signatures. Zero-phase filters were applied to ensure that there is no relative phase delay between decomposed signals. Results showed that a super-resolution image after frequency decomposition can be generated with three times lower number of acquisitions without sacrificing image quality.

Conference paper

Harput S, Christensen-Jeffries K, Li Y, Brown J, Eckersley RJ, Dunsby C, Tang M-Xet al., 2017, Two Stage Sub-Wavelength Motion Correction in Human Microvasculature for CEUS Imaging, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719

The structure of microvasculature cannot be resolved using clinical B-mode or contrast-enhanced ultrasound (CEUS) imaging due to the fundamental diffraction limit at clinical ultrasound frequencies. It is possible to overcome this resolution limitation by localizing individual microbubbles through multiple frames and forming a super-resolved image. However, ultrasound super-resolution creates its unique problems since the structures to be imaged are on the order of 10s of μm. Tissue movement much larger than 10 μm is common in clinical imaging, which can significantly reduce the accuracy of super-resolution images created from microbubble locations gathered through hundreds of frames. This study investigated an existing motion estimation algorithm from magnetic resonance imaging for ultrasound super-resolution imaging. Its correction accuracy is evaluated using simulations with increasing complexity of motion. Feasibility of the method for ultrasound super-resolution in vivo is demonstrated on clinical ultrasound images. For a chosen microvessel, the super-resolution image without motion correction achieved a sub-wavelength resolution; however after the application of proposed two-stage motion correction method the size of the vessel was reduced to half.

Conference paper

Brown J, Christensen-Jeffries K, Harput S, Dunsby C, Tang MX, Eckersley RJet al., 2017, Investigation of microbubble detection methods for super-resolution imaging of microvasculature, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719

Conference paper

Jeffries KC, Schirmer M, Brown J, Harput S, Tang MX, Dunsby C, Aljabar P, Eckersley Ret al., 2017, Automated super-resolution image processing in ultrasound using machine learning, ISSN: 1948-5719

© 2017 IEEE. Clinical implementation of super-resolution (SR) ultrasound imaging requires accurate single microbubble detection, and would benefit greatly from automation in order to minimize time requirements and user dependence. We present a machine learning based post-processing tool for the application of SR ultrasound imaging, where we utilize superpixelation and support vector machines (SVMs) for foreground detection and signal differentiation.

Conference paper

Jeffries K, Huang DY, Brown J, Harput S, Dunsby C, Tang MX, Sidhu PS, Eckersley Ret al., 2017, Super-resolution ultrasound to aid testicular lesion characterisation, ISSN: 1948-5719

© 2017 IEEE. Changes in microvascular structure and flow is of clinical importance in the study of a number of disease processes such as cancer and diabetes. Ultrasound is often the primary imaging procedure performed to determine appropriate treatment or surgery for testicular lesions. Currently, however, differentiation and diagnosis of both benign and malignant testicular tumours such as seminomas, leydig cell tumours and lymphomas are often challenging. Contrast-enhanced ultrasound (CEUS) has been used to aid their characterisation [1]. There are, however, a variety of benign testicular lesions that can mimic testicular malignancies. Ultrasound super-resolution (US-SR) techniques have been able to visualise vascular structures in vitro and in vivo beyond the diffraction limit by localizing individual microbubble signals. In this work, we aim to apply US-SR processing to clinical data to aid diagnostic confidence.

Conference paper

Brown J, Christensen-Jeffries K, Harput S, Dunsby C, Tang MX, Eckersley RJet al., 2017, Investigation of microbubble detection methods for super-resolution imaging of microvasculature, ISSN: 1948-5719

© 2017 IEEE. Super-resolution techniques that localise isolated bubble signals first require detection algorithms to separate the bubble and tissue responses. This work explores the available bubble detection techniques for super-resolution of tumour microvasculature. Pulse inversion (PI), differential imaging (DI) and singular value decomposition (SVD) filtering were compared in terms of the localisation accuracy, precision and contrast to tissue ratio (CTR). Bubble responses were simulated using the Marmottant model. Non-linear propagation through moving and stationary tissue was modelled using k-Wave. The results showed that PI signal was largely independent of flow direction and speed compared to SVD and DI which were less appropriate for lateral motion. At the lowest speeds, the bubble displacement between frames is not sufficient to generate a strong differential signal. SVD is unsuitable for stationary bubbles. For super-resolution of tumour microvasculature, the results suggest that non-linear techniques are preferential.

Conference paper

Jeffries KC, Harput S, Brown J, Dunsby C, Aljabar P, Tang MX, Eckersley Ret al., 2017, Microbubble localization errors in ultrasonic super-resolution imaging, ISSN: 1948-5719

© 2017 IEEE. Recently, acoustic super-resolution (SR) imaging has allowed visualization of microvascular structure and flow beyond the diffraction limit through the localization of many isolated microbubble signals. Each bubble position is typically estimated by calculating the centroid, finding a local maximum, or finding the peak of a 2-D Gaussian function fit. However, the backscattered signal from a microbubble depends not only on diffraction characteristics of the waveform, but also on the bubble behavior in the acoustic field, which if not accounted for, may cause localization errors.

Conference paper

Sherlock B, Warren SC, Alexandrov Y, Yu F, Stone J, Knight J, Neil MAA, Paterson C, French PMW, Dunsby CWet 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.

Journal article

Christensen-Jeffries K, Harput S, Brown J, Wells PNT, Aljabar P, Dunsby CW, Tang M, Eckersley RJet al., 2017, Microbubble Axial Localization Errors inUltrasound Super-Resolution Imaging, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 64, Pages: 1644-1654, ISSN: 0885-3010

Acoustic super-resolution imaging has allowed visualization of microvascular structure and flow beyond the diffraction limit using standard clinical ultrasound systems through the localization of many spatially isolated microbubble signals. The determination of each microbubble position is typically performed by calculating the centroid, finding a local maximum, or finding the peak of a 2-D Gaussian function fit to the signal. However, the backscattered signal from a microbubble depends not only on diffraction characteristics of the waveform, but also on the microbubble behavior in the acoustic field. Here, we propose a new axial localization method by identifying the onset of the backscattered signal. We compare the accuracy of localization methods using in vitro experiments performed at 7 cm depth and 2.3 MHz center frequency. We corroborate these findings with simulated results based on the Marmottant model. We show experimentally and in simulations that detecting the onset of the returning signal provides considerably increased accuracy for super-resolution. Resulting experimental cross-sectional profiles in super-resolution images demonstrate at least 5.8 times improvement in contrast ratio and more than 1.8 reduction in spatial spread (provided by 90% of the localizations) for the onset method over centroiding, peak detection and 2D Gaussian fitting methods. Simulations estimate that these latter methods could create errors in relative bubble positions as high as 900 μ m at these experimental settings, while the onset method reduced the interquartile range of these errors by a factor of over 2.2. Detecting the signal onset is therefore expected to considerably improve the accuracy of super-resolution.

Journal article

Christensen-Jeffries K, Brown J, Aljabar P, Tang M, Dunsby CW, Eckersley RJet al., 2017, 3-D In Vitro Acoustic Super-Resolution andSuper-Resolved Velocity Mapping UsingMicrobubbles, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 64, Pages: 1478-1486, ISSN: 0885-3010

Standard clinical ultrasound (US) imaging frequencies are unable to resolve microvascular structures due to the fundamental diffraction limit of US waves. Recent demonstrations of 2D super-resolution both in vitro and in vivo have demonstrated that fine vascular structures can be visualized using acoustic single bubble localization. Visualization of more complex and disordered 3D vasculature, such as that of a tumor, requires an acquisition strategy which can additionally localize bubbles in the elevational plane with high precision in order to generate super-resolution in all three dimensions. Furthermore, a particular challenge lies in the need to provide this level of visualization with minimal acquisition time. In this work, we develop a fast, coherent US imaging tool for microbubble localization in 3D using a pair of US transducers positioned at 90°. This allowed detection of point scatterer signals in 3 dimensions with average precisions equal to 1.9 µm in axial and elevational planes, and 11 µm in the lateral plane, compared to the diffraction limited point spread function full widths at half maximum of 488 µm, 1188 µm and 953 µm of the original imaging system with a single transducer. Visualization and velocity mapping of 3D in vitro structures was demonstrated far beyond the diffraction limit. The capability to measure the complete flow pattern of blood vessels associated with disease at depth would ultimately enable analysis of in vivo microvascular morphology, blood flow dynamics and occlusions resulting from disease states.

Journal article

Gorlitz F, Corcoran DS, Garcia Castano EA, Leitinger B, Neil MAA, Dunsby CW, French PMWet 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.

Journal article

Noble E, Kumar S, Gorlitz F, Stain C, Dunsby CW, French PMWet al., 2017, In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime, Plant Methods, Vol: 13, ISSN: 1746-4811

BackgroundIn order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe.ResultsOur studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For

Journal article

Lagarto J, Hares JD, Dunsby CW, French PMWet al., 2017, Development of low-cost instrumentation for single point autofluorescence lifetime measurements, Journal of Fluorescence, Vol: 27, Pages: 1643-1654, ISSN: 1573-4994

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.

Journal article

Sikkel MB, Kumar S, Maioli V, Rowlands C, Gordon F, Harding SE, Lyon AR, MacLeod KT, Dunsby Cet al., 2017, Erratum: High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes: [J. Biophotonics 9, No. 3, 311-323 (2016)]., J Biophotonics, Vol: 10, Pages: 744-745

In the article by M.B. Sikkel et al. (doi: 10.1002/jbio.201500193), published in J. Biophotonics 9, 311-323 (2016), an error occurred in the computer code that was used to generate Figure 3. This erratum is published to correct Figure 3, the calculated value of tgeom and the experimentally determined value of toptics in the text of the article.

Journal article

Sparks H, Gorlitz F, Kelly D, Warren SC, Kellet PA, Garcia E, Dymoke-Bradshaw AKL, Hares JD, Neil MAA, Dunsby C, French PMWet 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).

Journal article

French PMW, Görlitz F, Kelly D, Warren S, Alibhai D, West L, Kumar S, Alexandrov Y, Munro I, McGinty J, Talbot C, Serwa R, Thinon E, Da Paola V, Murray EJ, Stuhmeier F, Neil M, Tate E, Dunsby Cet 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.

Journal article

Perdios L, Lowe AR, Saladino G, Bunney TD, Thiyagarajan N, Alexandrov Y, Dunsby C, French PM, Chin JW, Gervasio FL, Tate EW, Katan Met al., 2017, Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET, Scientific Reports, Vol: 7, ISSN: 2045-2322

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

Journal article

Go¨rlitz F, Corcoran DS, Sparks H, Leitinger B, Neil M, Dunsby C, French Pet al., 2017, Mapping molecular function to biological nanostructure: Combining structured illumination microscopy with fluorescence lifetime imaging

© 2017 OSA. 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.

Conference paper

Maioli V, Chennell G, Sparks H, Lana T, Kumar S, Carling D, Sardini A, Dunsby CWet al., 2016, Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates, Scientific Reports, Vol: 6, ISSN: 2045-2322

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Journal article

Cortés E, Huidobro PA, Sinclair HG, Guldbrand S, Peveler WJ, Davies T, Parrinello S, Görlitz F, Dunsby C, Neil MAA, Sivan Y, Parkin IP, French PMW, Maier SAet 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.

Journal article

Margineanu A, Chan JJ, Kelly DJ, Warren SC, Flatters D, Kumar S, Katan M, Dunsby CW, French PMet al., 2016, Corrigendum: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)., Scientific Reports, Vol: 6, ISSN: 2045-2322

Journal article

Warren SC, Kim Y, Stone JM, Mitchell C, Knight JC, Neil MAA, Paterson C, French PMW, Dunsby CWet 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.

Journal article

Gore DM, French P, O'Brart D, Dunsby C, Allan BDSet al., 2016, NC-1059 peptide-assisted transepithelial riboflavin penetration in an ex-vivo rabbit corneal model, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

Chennell G, Willows RJW, Warren SC, Carling D, French PMW, Dunsby C, Sardini Aet al., 2016, Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM, Sensors, Vol: 16, ISSN: 1424-8239

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

Journal article

This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.

Request URL: http://wlsprd.imperial.ac.uk:80/respub/WEB-INF/jsp/search-html.jsp Request URI: /respub/WEB-INF/jsp/search-html.jsp Query String: limit=30&id=00321238&person=true&page=2&respub-action=search.html