270 results found
Hong W, Sparks H, Dunsby C, 2023, Alignment and Characterisation of Remote-RefocusingSystems, Applied Optics, ISSN: 1559-128X
Smith M, Sparks H, Almagro J, et al., 2023, Active mesh and neural network pipeline for cell aggregate segmentation, Biophysical Journal, Vol: 122, Pages: 1586-1599, ISSN: 0006-3495
Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.
Riemer K, Toulemonde M, Yan J, et al., 2023, Fast and selective super-resolution ultrasound in vivo with acoustically activated nanodroplets, IEEE Transactions on Medical Imaging, Vol: 42, Pages: 1056-1067, ISSN: 0278-0062
Perfusion by the microcirculation is key to the development, maintenance and pathology of tissue. Its measurement with high spatiotemporal resolution is consequently valuable but remains a challenge in deep tissue. Ultrasound Localization Microscopy (ULM) provides very high spatiotemporal resolution but the use of microbubbles requires low contrast agent concentrations, a long acquisition time, and gives little control over the spatial and temporal distribution of the microbubbles. The present study is the first to demonstrate Acoustic Wave Sparsely-Activated Localization Microscopy (AWSALM) and fast-AWSALM for in vivo super-resolution ultrasound imaging, offering contrast on demand and vascular selectivity. Three different formulations of acoustically activatable contrast agents were used. We demonstrate their use with ultrasound mechanical indices well within recommended safety limits to enable fast on-demand sparse activation and destruction at very high agent concentrations. We produce super-localization maps of the rabbit renal vasculature with acquisition times between 5.5 s and 0.25 s, and a 4-fold improvement in spatial resolution. We present the unique selectivity of AWSALM in visualizing specific vascular branches and downstream microvasculature, and we show super-localized kidney structures in systole (0.25 s) and diastole (0.25 s) with fast-AWSALM outdoing microbubble based ULM. In conclusion, we demonstrate the feasibility of fast and selective measurement of microvascular dynamics in vivo with subwavelength resolution using ultrasound and acoustically activatable nanodroplet contrast agents.
Dvinskikh L, Sparks H, Brito L, et al., 2023, Remote-refocusing light-sheet fluorescence microscopy enables 3D imaging of electromechanical coupling of hiPSC-derived and adult cardiomyocytes in co-culture, Scientific Reports, Vol: 13, Pages: 1-14, ISSN: 2045-2322
Improving cardiac function through stem-cell regenerative therapy requires functional and structural integration of the transplanted cells with the host tissue. Visualizing the electromechanical interaction between native and graft cells necessitates 3D imaging with high spatio-temporal resolution and low photo-toxicity. A custom light-sheet fluorescence microscope was used for volumetric imaging of calcium dynamics in co-cultures of adult rat left ventricle cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. Aberration-free remote refocus of the detection plane synchronously to the scanning of the light sheet along the detection axis enabled fast dual-channel 3D imaging at subcellular resolution without mechanical sample disturbance at up to 8 Hz over a ∼300 µm × 40 µm × 50 µm volume. The two cell types were found to undergo electrically stimulated and spontaneous synchronized calcium transients and contraction. Electromechanical coupling improved with co-culture duration, with 50% of adult-CM coupled after 24 h of co-culture, compared to 19% after 4 h (p = 0.0305). Immobilization with para-nitroblebbistatin did not prevent calcium transient synchronization, with 35% and 36% adult-CM coupled in control and treated samples respectively (p = 0.91), indicating that electrical coupling can be maintained independently of mechanotransduction.
Dvinskikh L, Sparks H, MacLeod K, et al., 2023, High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes, Frontiers in Physiology, Vol: 14, Pages: 1-14, ISSN: 1664-042X
Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity.Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 μm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell.Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules.Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure.
Hong W, Wright T, Sparks H, et al., 2022, Adaptive light-sheet fluorescence microscopy with a deformable mirror for video-rate volumetric imaging, Applied Physics Letters, Vol: 121, Pages: 1-7, ISSN: 0003-6951
Light-sheet fluorescence microscopy (LSFM) achieves optically sectioned imaging with the relatively low photobleaching and phototoxic effect. To achieve high-speed volumetric LSFM imaging without perturbing the sample, it is necessary to use some form of remote refocusing in the detection beam path. Previous work used electrically tunable lenses, tunable acoustic gradient index of refraction lenses, or the remote-refocusing approach of Botcherby et al. [Opt. Lett. 32(14), 2007 (2007)] to achieve remote refocusing. However, these approaches generally only provide low-order defocus correction, which is not compatible with higher-NA objectives that require higher order defocus corrections or reduce the optical throughput. In order to simultaneously achieve high-speed remote refocusing and correct system aberrations, we employ a deformable mirror in the detection path that is capable of providing higher orders of defocus and aberration correction in an optical system with an NA of 0.72–0.75. We demonstrate high-speed volumetric imaging at 26.3 volumes per second and 35 frames per volume for a defocus range of −50 to 50 μm.
Tang M, 2022, Super-resolution ultrasound localization microscopy of microvascular structure and flow for distinguishing metastatic lymph nodes – an initial human study, Ultraschall in der Medizin, Vol: 43, Pages: 592-598, ISSN: 0172-4614
Purpose Detecting and distinguishing metastatic lymph nodes (LNs) from those with benign lymphadenopathy are crucial for cancer diagnosis and prognosis but remain a clinical challenge. A recent advance in super-resolution ultrasound (SRUS) through localizing individual microbubbles has broken the diffraction limit and tracking enabled in vivo noninvasive imaging of vascular morphology and flow dynamics at a microscopic level. In this study we hypothesize that SRUS enables quantitative markers to distinguish metastatic LNs from benign ones in patients with lymphadenopathy.Materials and Methods Clinical contrast-enhanced ultrasound image sequences of LNs from 6 patients with lymph node metastasis and 4 with benign lymphadenopathy were acquired and motion-corrected. These were then used to generate super-resolution microvascular images and super-resolved velocity maps. From these SRUS images, morphological and functional measures were obtained including micro-vessel density, fractal dimension, mean flow speed, and Local Flow Direction Irregularity (LFDI) measuring the variance in local flow direction. These measures were compared between pathologically proven reactive and metastasis LNs.Results Our initial results indicate that the difference in the indicator of flow irregularity (LFDI) derived from the SRUS images is statistically significant between the two groups. The LFDI is 60% higher in metastatic LNs compared with reactive nodes.Conclusion This pilot study demonstrates the feasibility of super-resolution ultrasound for clinical imaging of lymph nodes and the potential of using the irregularity of local blood flow directions afforded by SRUS for the characterization of LNs.
Wysoczanski R, Baker JR, Fenwick P, et al., 2022, Defective Phagocytosis in COPD Macrophages Is Improved by Mitochondrial Antioxidants Without Alteration in Mitochondrial Function, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Hong W, Dunsby C, 2022, Automatic tube lens design from stock optics for microscope remote-refocusing systems, Optics Express, Vol: 30, Pages: 4274-4278, ISSN: 1094-4087
The remote-refocusing approach of Botcherby et al. [Opt. Lett. 32, 2007 (2007) [CrossRef] ] has been applied widely to 2D and 3D fluorescence microscopes to enable rapid refocusing of the optical system without mechanically perturbing the sample. In order for this approach to operate correctly, it requires that the overall magnification of the first two microscope systems matches the ratio of the refractive indices in sample and intermedia image spaces. However, commercially available tube lenses are not always suitable to produce the desired overall magnification. Therefore, a practical approach to produce tube lenses with low expense and diffraction-limited performance is required. Tube lenses can be formed using a pair of stock achromatic doublets, however, selecting appropriate pairs of achromatic doublets from stock optics is a time-consuming process, as many combinations can be considered. In this paper, we present two software packages (Catalogue Generator and Doublet Selector) developed in MATLAB that use the application programming interface (ZOS-API) to the Zemax OpticStudio optical design software to realise an automatic search of stock achromatic doublets to produce microscope tube lenses with a specified focal length, entrance pupil diameter and maximum design field angle. An algorithm to optimise principal plane positions in versions of OpticStudio before 20.2 was also introduced to enable the use of older software versions. To evaluate the performance of Catalogue Generator and Doublet Selector, we used them to generate ten tube lens designs. All of the software-produced tube lenses have a better optical performance than those using manually selected pairs of stock doublets lenses.
Ramuz M, Diakonov I, Dunsby C, et al., 2022, MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET., Methods Mol Biol, Vol: 2483, Pages: 33-59
The newly generated software plugin MultiFRET allows for real-time measurements of multiplexed fluorescent biosensors in a near high-throughput fashion. Here we describe a detailed protocol for setup and use of this software for any purpose requiring instant feedback during fluorescence measurement experiments. We further describe its non-primary features including beam splitter misalignment correction, custom calculations through input of simple equations typed in a .txt format, customizable Excel output, and offline bulk analysis of image stacks. Finally, we supply a usage example of a cAMP measurement in cultured rat neonatal cardiomyocytes.
Wright T, Sparks H, Paterson C, et al., 2021, Video-rate remote refocusing through continuous oscillation of a membrane deformable mirror, Journal of Physics: Photonics, Vol: 3, Pages: 1-14, ISSN: 2515-7647
This paper presents the use of a deformable mirror (DM) configured to rapidly refocus a microscope employing a highnumerical aperture objective lens. An Alpao DM97-15 membrane DM was used to refocus a 40×/0.80 NA water-immersionobjective through a defocus range of -50 to 50 m at 26.3 sweeps per second. We achieved imaging with a mean Strehlmetric of > 0.6 over a field of view in the sample of 200×200 m2 over a defocus range of 77 m. We describe anoptimisation procedure where the mirror is swept continuously in order to avoid known problems of hysteresis associatedwith the membrane DM employed. This work demonstrates that a DM-based refocusing system could in the future be used inlight-sheet fluorescence microscopes to achieve video-rate volumetric imaging.
Kalita R, Flanagan W, Lightley J, et al., 2021, Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast, JOURNAL OF BIOPHOTONICS, Vol: 14, ISSN: 1864-063X
Wysoczanski R, Baker J, Fenwick P, et al., 2021, Image analysis of tissue macrophages to confirm differential phagocytosis between groups by microscopy and automated bacterial quantification, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Lightley J, Gorlitz F, Kumar S, et al., 2021, Robust deep learning optical autofocus system applied to automated multiwell plate single molecule localization microscopy, JOURNAL OF MICROSCOPY, ISSN: 0022-2720
Garcia E, Lightley J, Kumar S, et al., 2021, Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease, Publisher: SPRINGER, Pages: S142-S142, ISSN: 0945-6317
Garcia E, Lightley J, Kumar S, et al., 2021, Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease, JOURNAL OF PATHOLOGY CLINICAL RESEARCH, Vol: 7, Pages: 438-445
Cannon T, Lagarto J, Dyer B, et al., 2021, Characterisation of NADH fluorescence properties under one-photon excitation with respect to temperature, pH and binding to lactate dehydrogenase, Optical Society of America Continuum, Vol: 4, Pages: 1610-1625
Reduced nicotinamide adenine dinucleotide (NADH) is the principal electron donor in glycolysis and oxidative metabolism and is thus recognized as a key biomarker for probing metabolic state. While the fluorescence characteristics of NADH have been investigated extensively, there are discrepancies in the published data due to diverse experimental conditions, instrumentation and microenvironmental parameters that can affect NADH fluorescence. Using a cuvette-based time-resolved spectrofluorimeter employing one-photon excitation at 375 nm, we characterized the fluorescence intensity, lifetime, spectral response, anisotropy and time-resolved anisotropy of NADH in aqueous solution under varying microenvironmental conditions, namely temperature, pH, and binding to lactate dehydrogenase (LDH). Our results demonstrate how temperature, pH, and binding partners each impact the fluorescence signature of NADH and highlight the complexity of the fluorescence data when different parameters produce competing effects. We hope that the data presented in this study will provide a reference for potential sources of variation in experiments measuring NADH fluorescence.
Dvinskikh L, Harding S, Sparks H, et al., 2021, High speed imaging of calcium dynamics in cardiomyocytes with a flexible light-sheet fluorescence microscope, Biophotonics Congress 2021
Kondo H, Ratcliffe CDH, Hooper S, et al., 2021, Single-cell resolved imaging reveals intra-tumor heterogeneity in glycolysis, transitions between metabolic states, and their regulatory mechanisms, CELL REPORTS, Vol: 34, ISSN: 2211-1247
Jones DC, Alexandrov Y, Curry N, et al., 2021, Multidimensional spectroscopy and imaging of defects in synthetic diamond: excitation-emission-lifetime luminescence measurements with multiexponential fitting and phasor analysis, Journal of Physics D: Applied Physics, Vol: 54, Pages: 1-13, ISSN: 0022-3727
We report the application of phasor analysis and nonlinear iterative fitting to complex spatial and spectroscopic luminescence decay data obtained from multidimensional microscopy of a CVD diamond grown on a HPHT substrate. This spectral and lifetime-resolved analysis enabled spatial mapping of variations in concentrations of nitrogen vacancy (NV) defects in both charge states and the quenching of NV− defects, as well as the identification of SiV− luminescence. These imaging and spectroscopic modalities may be important for reliable fabrication of quantum devices based on such defects in diamond, which will require well-defined and characterised quantum electronic properties.
Lightley J, Görlitz F, Kumar S, et al., 2021, ROBUST OPTICAL AUTOFOCUS SYSTEM UTILIZING NEURAL NETWORKS APPLIED TO AUTOMATED MULTIWELL PLATE STORM MICROSCOPY, ISSN: 1605-7422
We present a robust, low-cost neural network-based optical autofocus system that can operate over a range of ±100µm with submicron precision, enabling automated high-content super-resolved imaging with a 1.3 NA objective lens.
Sparks H, Almagro J, Behrens A, et al., 2021, Dual-view oblique plane microscopy
Dual-view Oblique Plane Microscopy (dOPM) enables single-objective multi-view light-sheet fluorescence microscopy. This talk introduces the dOPM concept and demonstrates optical resolution performance with exemplar 3D datasets of fluorescence bead samples and fixed multicellular spheroids.
Lightley J, Gorlitz F, Kumar S, et al., 2021, ROBUST OPTICAL AUTOFOCUS SYSTEM UTILIZING NEURAL NETWORKS APPLIED TO AUTOMATED MULTIWELL PLATE STORM MICROSCOPY, European Conferences on Biomedical Optics - Advances in Microscopic Imaging III, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Kalita R, Lightley J, Kumar S, et al., 2021, Single-shot quantitative phase contrast using polarisation-resolved differential phase microscopy, European Conferences on Biomedical Optics - Advances in Microscopic Imaging III, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Kalita R, Lightley J, Kumar S, et al., 2021, Single-shot quantitative phase contrast using polarisation-resolved differential phase microscopy, ISSN: 1605-7422
We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated.
Jones B, McGlone ER, Fang Z, et al., 2021, Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signalling at glucagon family receptors, Journal of Biological Chemistry, Vol: 296, Pages: 1-15, ISSN: 0021-9258
Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR) and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitisation and downregulation due to recruitment of β-arrestins. Indeed, recently described GLP-1R agonists with reduced β-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both β-arrestin isoforms the duration of G protein-dependent cAMP/PKA signalling was increased in response to the endogenous ligand for each receptor. Moreover, in wild-type cells, “biased” GLP-1, GCG and GIP analogues with selective reductions in β-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogues increased the duration of cAMP signalling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for development of GLP-1R, GIPR and GCGR agonists with reduced β-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.
We present a new folded dual-view oblique plane microscopy (OPM) techniquetermed dOPM that enables two orthogonal views of the sample to be obtained by translating apair of tilted mirrors in refocussing space. Using a water immersion 40× 1.15 NA primaryobjective, deconvolved image volumes of 200 nm beads were measured to have full width athalf maxima (FWHM) of 0.35±0.04 μm and 0.39±0.02 μm laterally and 0.81±0.07 μm axially.The measured z-sectioning value was 1.33±0.45 μm using light-sheet FWHM in the frames ofthe two views of 4.99±0.58 μm and 4.89±0.63 μm. To qualitatively demonstrate that the systemcan reduce shadow artefacts while providing a more isotropic resolution, a multi-cellularspheroid approximately 100 μm in diameter was imaged.
Wysoczanski R, Baker JR, Fenwick P, et al., 2020, Analysis of defective phagocytosis in COPD using super-resolution microscopy and automated bacterial quantification, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Sparks H, Dvinskikh L, Firth J, et al., 2020, Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure, Journal of Biophotonics, Vol: 13, ISSN: 1864-063X
We report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.
Fang Z, Chen S, Pickford P, et al., 2020, The influence of peptide context on signaling and trafficking of glucagon-like peptide-1 receptor biased agonists, ACS Pharmacology & Translational Science, Vol: 3, Pages: 345-360, ISSN: 2575-9108
Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated cyclic AMP signaling, recruitment of β-arrestins, endocytosis, and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific midpeptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level.
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