Imperial College London

Dr Chris Dunsby

Faculty of Natural SciencesDepartment of Physics

Reader in Biomedical Optics
 
 
 
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Contact

 

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

 
 
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Location

 

622Blackett LaboratorySouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

223 results found

Ramuz M, Hasan A, Gruscheski L, Diakonov I, Pavlaki N, Nikolaev VO, Harding S, Dunsby C, Gorelik Jet al., 2019, A software tool for high-throughput real-time measurement of intensity-based ratio-metric FRET, Cells, Vol: 8, ISSN: 2073-4409

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.

Journal article

Corcoran D, Juskaite V, Xu Y, Gorlitz F, Alexandrov Y, Dunsby C, French P, Leitinger Bet al., 2019, DDR1 autophosphorylation is a result of aggregation into dense clusters, Scientific Reports, Vol: 9, ISSN: 2045-2322

The collagen receptor DDR1 is a receptor tyrosine kinase that promotes progression ofa wide range of human disorders. Little is known about how ligand binding triggers DDR1 kinase activity. We previously reported that collagen induces DDR1 activation through lateral dimer association and phosphorylation between dimers, a process that requires specific transmembrane association. Here we demonstrate ligand-induced DDR1 clustering by widefield and super-resolution imaging and provide evidence for a mechanism whereby DDR1 kinase activity is determined by its molecular density. Ligand binding resulted in initial DDR1 reorganisation into morphologically distinct clusters with unphosphorylated DDR1. Further compaction over time led to clusters with highly aggregated and phosphorylated DDR1. Ligand-induced DDR1 clustering was abolished by transmembrane mutations but did not require kinase activity. Our results significantly advance our understanding of the molecular events underpinning ligand-induced DDR1 kinase activity and provide an explanation for the unusually slow DDR1 activation kinetics.

Journal article

Jones DC, Kumar S, Lanigan PMP, McGuinness CD, Dale MW, Twitchen DJ, Fisher D, Martineau PM, Neil M, Dunsby C, French Pet 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.

Journal article

Harput S, Christensen-Jeffries K, Ramalli A, Brown J, Zhu J, Zhang G, Leow C, Toulemond M, Boni E, Tortoli P, Eckersley R, Dunsby C, Tang M-Xet al., 3-D super-resolution ultrasound imaging with a 2-D sparse array, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, ISSN: 0885-3010

High frame rate 3-D ultrasound imaging technologycombined with super-resolution processing method can visualize3-D microvascular structures by overcoming the diffractionlimited resolution in every spatial direction. However, 3-D superresolution ultrasound imaging using a full 2-D array requires asystem with large number of independent channels, the designof which might be impractical due to the high cost, complexity,and volume of data produced.In this study, a 2-D sparse array was designed and fabricatedwith 512 elements chosen from a density-tapered 2-D spirallayout. High frame rate volumetric imaging was performed usingtwo synchronized ULA-OP 256 research scanners. Volumetricimages were constructed by coherently compounding 9-angleplane waves acquired at a pulse repetition frequency of 4500Hz. Localization-based 3-D super-resolution images of two touching sub-wavelength tubes were generated from 6000 volumesacquired in 12 seconds. In conclusion, this work demonstratesthe feasibility of 3-D super-resolution imaging and super-resolvedvelocity mapping using a customized 2-D sparse array transducer

Journal article

Christensen-Jeffries K, Brown J, Harput S, Zhang G, Zhu J, Tang M-X, Dunsby C, Eckersley RJet al., 2019, Poisson statistical model of ultrasound super-resolution imaging acquisition time, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 66, Pages: 1246-1254, ISSN: 0885-3010

A number of acoustic super-resolution techniques have recently been developed to visualize microvascular structure and flow beyond the diffraction limit. A crucial aspect of all ultrasound (US) super-resolution (SR) methods using single microbubble localization is time-efficient detection of individual bubble signals. Due to the need for bubbles to circulate through the vasculature during acquisition, slow flows associated with the microcirculation limit the minimum acquisition time needed to obtain adequate spatial information. Here, a model is developed to investigate the combined effects of imaging parameters, bubble signal density, and vascular flow on SR image acquisition time. We find that the estimated minimum time needed for SR increases for slower blood velocities and greater resolution improvement. To improve SR from a resolution of λ/10 to λ/20 while imaging the microvasculature structure modeled here, the estimated minimum acquisition time increases by a factor of 14. The maximum useful imaging frame rate to provide new spatial information in each image is set by the bubble velocity at low blood flows (<;150 mm/s for a depth of 5 cm) and by the acoustic wave velocity at higher bubble velocities. Furthermore, the image acquisition procedure, transmit frequency, localization precision, and desired super-resolved image contrast together determine the optimal acquisition time achievable for fixed flow velocity. Exploring the effects of both system parameters and details of the target vasculature can allow a better choice of acquisition settings and provide improved understanding of the completeness of SR information.

Journal article

Lagarto J, Dyer B, Dunsby C, Peters N, French P, Dunsby C, Lyon Aet al., 2019, In vivo label-free optical monitoring of structural and metabolic remodeling of myocardium following infarction, Biomedical Optics Express, Vol: 10, Pages: 3506-3521, ISSN: 2156-7085

Cardiac remodeling following myocardial infarction (MI) involves structural and functional alterations in the infarcted and remote viable myocardium that can ultimately lead to heart failure. The underlying mechanisms are not fully understood and, following our previous study of the autofluorescence lifetime and diffuse reflectance signatures of the myocardium in vivo at 16 weeks post MI in rats [Biomed. Opt. Express 6(2), 324 (2015)], we here present data obtained at 1, 2 and 4 weeks post myocardial infarction that help follow the temporal progression of these changes. Our results demonstrate that both structural and metabolic changes in the heart can be monitored from the earliest time points following MI using label-free optical readouts, not only in the region of infarction but also in the remote non-infarcted myocardium. Changes in the autofluorescence intensity and lifetime parameters associated with collagen type I autofluorescence were indicative of progressive collagen deposition in tissue that was most pronounced at earlier time points and in the region of infarction. In addition to significant collagen deposition in infarcted and non-infarcted myocardium, we also report changes in the autofluorescence parameters associated with reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD), which we associate with metabolic alterations throughout the heart. Parallel measurements of the diffuse reflectance spectra indicated an increased contribution of reduced cytochrome c. Our findings suggest that combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy could provide a useful means to monitor cardiac function in vivo at the time of surgery.

Journal article

Guo W, Kumar S, Gorlitz F, garcia EC, Alexandrov Y, Munro I, Kelly D, Warren S, Thorpe P, Dunsby C, French Pet al., 2019, Automated fluorescence lifetime imaging high content analysis of Förster resonance energy transfer between endogenously-labeled kinetochore proteins in live budding yeast cells, Slas Technology, Vol: 24, Pages: 308-320, ISSN: 2472-6303

We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.

Journal article

Zhu J, Rowland E, Harput S, Riemer K, Leow CH, Clark B, Cox K, Lim A, Christensen-Jeffries K, Zhang G, Brown J, Dunsby C, Eckersley R, Weinberg P, Tang Met al., 2019, 3D super-resolution ultrasound imaging of rabbit lymph node vasculature in vivo using microbubbles, Radiology, Vol: 291, Pages: 642-650, ISSN: 0033-8419

Background: Variations in lymph node (LN) microcirculation can be indicative of metastasis. Identifying and quantifying metastatic LNs remains essential for prognosis and treatment planning but a reliable non-invasive imaging technique is lacking. 3D super-resolution (SR) ultrasound has shown potential to noninvasively visualize microvascular networks in vivo.Purpose: To study the feasibility of 3D SR ultrasound imaging of rabbit lymph node (LN) microvascular structure and blood flow using microbubbles.Materials and Methods: In vivo studies were carried out to image popliteal LNs of two healthy male New Zealand White rabbits aged 6-8 weeks. 3D high frame rate contrast enhanced ultrasound was achieved by mechanically scanning a linear imaging probe. Individual microbubbles were identified, localized, and tracked to form 3D SR images and super-resolved velocity maps. Acoustic sub-aperture processing (ASAP)was used to improve image contrast and generateenhanced power Doppler (PD) and color Doppler (CD) images. Vessel size and blood flow velocity distributions were evaluated and assessed by Student’s paired t-test. Results:SR images revealed micro-vessels in the rabbitLN, with branches clearly resolved when separated by 30 μm, which is less than half of the acoustic wavelength and not resolvable by power or color Doppler. The apparent size distribution of most vessels in the SR images was below 80 μm and agrees with micro-CT data whereas most of those detected by Doppler techniques were larger than 80 μm. The blood flow velocity distribution indicated that most of the blood flow in the rabbit popliteal LN was at velocities lower than 5mm/s. Conclusion: 3D super-resolution ultrasound imaging using microbubbles allows non-invasive and non-ionizing visualization and quantification of rabbit lymph node microvascular structures and blood flow dynamics with resolution below the wave diffraction limit.

Journal article

Zhang G, Harput S, Hu H, Christensen-Jeffries K, Zhu J, Brown J, Leow CH, Eckersley R, Dunsby C, Tang M-Xet al., 2019, Fast acoustic wave sparsely activated localization microscopy (fast-AWSALM): ultrasound super-resolution using plane-wave activation of nanodroplets, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 66, Pages: 1039-1046, ISSN: 0885-3010

Localization-based ultrasound super-resolution imaging using microbubble contrast agents and phase-change nano-droplets has been developed to visualize microvascular structures beyond the diffraction limit. However, the long data acquisition time makes the clinical translation more challenging. In this study, fast acoustic wave sparsely activated localization microscopy (fast-AWSALM) was developed to achieve super-resolved frames with sub-second temporal resolution, by using low-boiling-point octafluoropropane nanodroplets and high frame rate plane waves for activation, destruction, as well as imaging. Fast-AWSALM was demonstrated on an in vitro microvascular phantom to super-resolve structures that could not be resolved by conventional B-mode imaging. The effects of the temperature and mechanical index on fast-AWSALM was investigated. Experimental results show that sub-wavelength micro-structures as small as 190 lm were resolvable in 200 ms with plane-wave transmission at a center frequency of 3.5 MHz and a pulse repetition frequency of 5000 Hz. This is about a 3.5 fold reduction in point spread function full-width-half-maximum compared to that measured in conventional B-mode, and two orders of magnitude faster than the recently reported AWSALM under a non-flow/very slow flow situations and other localization based methods. Just as in AWSALM, fast-AWSALM does not require flow, as is required by current microbubble based ultrasound super resolution techniques. In conclusion, this study shows the promise of fast-AWSALM, a super-resolution ultrasound technique using nanodroplets, which can generate super-resolution images in milli-seconds and does not require flow.

Journal article

Brown J, Christensen-Jeffries K, Harput S, Zhang G, Zhu J, Dunsby C, Tang M-X, Eckersley RJet al., 2019, Investigation of microbubble detection methods for super-resolution imaging of microvasculature, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 66, Pages: 676-691, ISSN: 0885-3010

Ultrasound super-resolution techniques use the response of microbubble contrast agents (MBs) to visualize the microvasculature. Techniques that localize isolated bubble signals first require detection algorithms to separate the MB and tissue responses. This work explores the three main MB detection techniques for super-resolution of microvasculature. Pulse inversion (PI), differential imaging (DI) and singular value decomposition (SVD) filtering were compared in terms of the localization accuracy, precision and contrast to tissue ratio (CTR). MB responses were simulated based on the properties of Sonovue™ and using the Marmottant model. Non-linear propagation through tissue was modelled using the k-Wave software package. For the parameters studied, the results show that PI is most appropriate for low frequency applications, but also most dependent on transducer bandwidth. SVD is preferable for high frequency acquisition where localization precision on the order of a few microns is possible. PI is largely independent of flow direction and speed compared to SVD and DI, so is appropriate for visualizing the slowest flows and tortuous vasculature. SVD is unsuitable for stationary MBs and can introduce a localization error on the order of hundreds of microns over the speed range 0- 2 mm/s and flow directions from lateral (parallel to probe) to axial (perpendicular to probe). DI is only suitable for flow rates > 0.5 mm/s or as flow becomes more axial. Overall, this study develops a MB and tissue non-linear simulation platform to improve understanding of how different MB detection techniques can impact the super-resolution process and explores some of the factors influencing the suitability of each.

Journal article

Harput S, Christensen-Jeffries K, Ramalli A, Brown J, Zhu J, Zhang G, Leow CH, Toulemonde M, Boni E, Tortoli P, Eckersley RJ, Dunsby C, Tang M-Xet al., 2019, 3-D super-resolution ultrasound (SR-US) imaging with a 2-D sparse array

High frame rate 3-D ultrasound imaging technology combined withsuper-resolution processing method can visualize 3-D microvascular structuresby overcoming the diffraction limited resolution in every spatial direction.However, 3-D super-resolution ultrasound imaging using a full 2-D arrayrequires a system with large number of independent channels, the design ofwhich might be impractical due to the high cost, complexity, and volume of dataproduced. In this study, a 2-D sparse array was designed and fabricated with 512elements chosen from a density-tapered 2-D spiral layout. High frame ratevolumetric imaging was performed using two synchronized ULA-OP 256 researchscanners. Volumetric images were constructed by coherently compounding 9-angleplane waves acquired in 3 milliseconds at a pulse repetition frequency of 3000Hz. To allow microbubbles sufficient time to move between consequent compoundedvolumetric frames, a 7-millisecond delay was introduced after each volumeacquisition. This reduced the effective volume acquisition speed to 100 Hz andthe total acquired data size by 3.3-fold. Localization-based 3-Dsuper-resolution images of two touching sub-wavelength tubes were generatedfrom 6000 volumes acquired in 60 seconds. In conclusion, this work demonstratesthe feasibility of 3D super-resolution imaging and super-resolved velocitymapping using a customized 2D sparse array transducer.

Working paper

Munro I, Garcia EAC, Yan M, Guldbrand S, Kumar S, Kwakwa K, Dunsby C, Neil M, French Pet 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.

Journal article

Görlitz F, Lightley J, Kumar S, Garcia E, Yan M, Wysoczanski R, Alexandrov Y, Baker JR, Barnes PJ, Munro I, Donnelly LE, Dunsby C, Neil MAA, French PMWet al., 2019, Automated multiwell plate STORM: Towards open source super-resolved high content analysis, ISSN: 1605-7422

© 2019 SPIE. Among super-resolved microscopy (SRM) methods, single molecule localisation microscopy techniques, such as photo-activated localisation microscopy (PALM) [1] and stochastic optical reconstruction microscopy (STORM) [2], enable imaging beyond the classical diffraction limit to gain new insights in subcellular biological processes with relatively simple instrumentation. This has led to a number of low-cost instruments, e.g. for STORM microscopy [3-6], which can benefit from an array of software tools for the single molecule localisation microscopy (SMLM) data analysis [7]. Our low-cost "easySTORM" approach [4] implements dSTORM [8] with multimode diode lasers and optical fibres to provide STORM images with fields of view up to ∼125 μm diameter using μManager [9] to control the image data acquisition and ThunderSTORM [10] to analyse the SMLM data. We and others [11,12] are motivated to develop automated SMLM for high content analysis (HCA) that enable rapid imaging of sample arrays, allows statistical analysis of samples that may vary in terms of labelling and biological heterogeneity and enable moderate throughput screening applications.

Conference paper

Brown J, Kolas S, Christensen-Jeffries K, De Menczes C, Harput S, Zhu J, Zhang G, Tang M-X, Dunsby C, Eckersley RJet al., 2018, Development of Simultaneous Optical Imaging and Super-Resolution Ultrasound to Improve Microbubble Localization Accuracy, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719

Conference paper

Zhang G, Harput S, Hu H, Christensen-Jeffries K, Zhu J, Brown J, Leow CH, Dunsby C, Eckersley RJ, Tang M-Xet al., 2018, Fast Acoustic Wave Sparsely Activated Localization Microscopy (fast-AWSALM) using Octafluoropropane Nanodroplets, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719

Conference paper

Harput S, Christensen-Jeffries K, Brown J, Zhu J, Zhang G, Leow CH, Toulemonde M, Ramalli A, Boni E, Tortoli P, Eckersley RJ, Dunsby C, Tang Met al., 2018, 3-D super-resolution ultrasound (SR-US) imaging using a 2-D sparse array with high volumetric imaging rate, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE

Super-resolution ultrasound imaging has been sofar achieved in 3-D by mechanically scanning a volume witha linear probe, by co-aligning multiple linear probes, by usingmultiplexed 3-D clinical ultrasound systems, or by using 3-D ultrasound research systems. In this study, a 2-D sparsearray was designed with 512 elements according to a density-tapered 2-D spiral layout and optimized to reduce the sidelobesof the transmitted beam profile. High frame rate volumetricimaging with compounded plane waves was performed usingtwo synchronized ULA-OP256 systems. Localization-based 3-Dsuper-resolution images of two touching sub-wavelength tubeswere generated from a 120 second acquisition.

Conference paper

Harput S, Christensen-Jeffries K, Brown J, Zhu J, Zhang G, Eckersley RJ, Dunsby C, Tang Met al., 2018, 3-D motion correction for volumetric super-resolution ultrasound (SR-US) imaging, IEEE International Ultrasonics Symposium (IUS) 2018, Publisher: IEEE

Motion during image acquisition can cause imagedegradation in all medical imaging modalities. This is particularlyrelevant in 2-D ultrasound imaging, since out-of-plane motioncan only be compensated for movements smaller than elevationalbeamwidth of the transducer. Localization based super-resolutionimaging creates even a more challenging motion correction taskdue to the requirement of a high number of acquisitions to forma single super-resolved frame.In this study, an extension of two-stage motion correctionmethod is proposed for 3-D motion correction. Motion estimationwas performed on high volumetric rate ultrasound acquisitionswith a handheld probe. The capability of the proposed methodwas demonstrated with a 3-D microvascular flow simulation tocompensate for handheld probe motion. Results showed that two-stage motion correction method reduced the average localizationerror from 136 to 18μm.

Conference paper

Christensen-Jeffries K, Harput S, Brown J, Zhang G, Zhu J, Tang M-X, Dunsby C, Eckersley Ret al., 2018, 3D In Vitro Ultrasound Super-Resolution Imaging using a Clinical System, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719

Conference paper

Gorlitz F, Guldbrand S, Runcorn T, Murray R, Jaso-Tamame A, Sinclair H, Martinez-Perez E, Taylor J, Neil M, Dunsby CW, French Pet 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.

Journal article

Lagarto J, Dyer B, Talbot C, Peters N, French P, Lyon A, Dunsby Cet al., 2018, Characterization of NAD(P)H and FAD autofluorescence signatures in a Langendorff isolated-perfused rat heart model, Biomedical Optics Express, Vol: 9, Pages: 4978-4978, ISSN: 2156-7085

Autofluorescence spectroscopy is a promising label-free approach to characterize biological samples with demonstrated potential to report structural and biochemical alterations in tissues in a number of clinical applications. We report a characterization of the ex vivo autofluorescence fingerprint of cardiac tissue, exploiting a Langendorff-perfused isolated rat heart model to induce physiological insults to the heart, with a view to understanding how metabolic alterations affect the autofluorescence signals. Changes in the autofluorescence intensity and lifetime signatures associated with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized during oxygen- or glucose-depletion protocols. Results suggest that both NAD(P)H and FAD autofluorescence intensity and lifetime parameters are sensitive to changes in the metabolic state of the heart owing to oxygen deprivation. We also observed changes in NAD(P)H fluorescence intensity and FAD lifetime parameter on reperfusion of oxygen, which might provide information on reperfusion injury, and permanent tissue damage or changes to the tissue during recovery from oxygen deprivation. We found that changes in the autofluorescence signature following glucose-depletion are, in general, less pronounced, and most clearly visible in NAD(P)H related parameters. Overall, the results reported in this investigation can serve as baseline for future investigations of cardiac tissue involving autofluorescence measurements.

Journal article

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

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