The Experts below are selected from a list of 2748 Experts worldwide ranked by ideXlab platform
James Mcginty - One of the best experts on this subject based on the ideXlab platform.
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single shot Optical Projection Tomography for high speed volumetric imaging
bioRxiv, 2021Co-Authors: Connor Darling, James Mcginty, Sunil Kumar, Samuel P X Davis, P M W FrenchAbstract:We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of Projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 Projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under {pound}5,000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.
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exploiting patterned illumination and detection in Optical Projection Tomography conference presentation
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 2019Co-Authors: Samuel P X Davis, Teresa Correia, Laurence Bugeon, Paul Frankel, Marie-christine Ramel, Sunil Kumar, Laura Wisniewski, Simon R Arridge, Maggie Dallman, James McgintyAbstract:Optical Projection Tomography (OPT), the Optical equivalent of x-ray computed Tomography, reconstructs the 3D structure of a sample from a series of wide-field 2D Projections acquired at different angles [1]. OPT is used to map the Optical attenuation and/or fluorescence distributions of intact transparent samples without the need for mechanical sectioning. While it is typically applied to chemically cleared samples, it can also be used to image inherently transparent or weakly scattering live organisms including adult zebrafish up to ~1cm in diameter [2]. When applying OPT to live samples it is important to minimise the data acquisition time while maximising the image quality in the presence of scattering. The former issue can be addressed using compressive sensing to reduce the number of Projections required [3]. Scattered light can be rejected using structured illumination [4], but this removes emission from regions the excitation modulation does not reach and reduces the available dynamic range. To address this, we have explored the rejection of scattered light by acquiring Projections with parallel semi-confocal line illumination and detection in an approach we describe as slice-OPT (sl-OPT). The impact of Optical scattering can also be reduced by imaging at longer wavelengths [5]. We are exploring OPT in the NIR 1&2 spectral windows. However, exotic array detectors, e.g. for short wave infrared light, are costly and so we are also developing a single pixel camera [6] approach. We will present our progress applying these techniques to 3D imaging of vasculature and tumour burden in live adult zebrafish. [1] Sharpe et al, Science, vol. 296, Issue 5567, pp. 541-545, 2002. [2] Kumar et al, Oncotarget, vol. 7, no.28, pp. 43939-43948, 2016. [3] Correia et al, PloS one, vol. 10, no. 8, p. e0136213, 2015. [4] Kristensson et al, Optics express, vol. 20, no. 13, pp. 14437-14450, 2012. [5] Shi et al., Journal of Biophotonics, vol. 9, no. 1-2, pp. 38-43, 2016. [6] Duarte et al., IEEE Signal Processing Magazine, vol. 25, no. 2, pp. 83-91, 2008.
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slice illuminated Optical Projection Tomography
Optics Letters, 2018Co-Authors: Samuel P X Davis, Teresa Correia, Paul Frankel, James Mcginty, Sunil Kumar, Laura Wisniewski, Simon R Arridge, Paul M. W. FrenchAbstract:To improve the imaging performance of Optical Projection Tomography (OPT) in live samples, we have explored a parallelized implementation of semi-confocal line illumination and detection to discriminate against scattered photons. Slice-illuminated OPT (sl-OPT) improves reconstruction quality in scattering samples by reducing interpixel crosstalk at the cost of increased acquisition time. For in vivo imaging, this can be ameliorated through the use of compressed sensing on angularly undersampled OPT data sets. Here, we demonstrate sl-OPT applied to 3D imaging of bead phantoms and live adult zebrafish.
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functional imaging of live zebrafish using fluorescence lifetime Optical Projection Tomography conference presentation
Proceedings of SPIE, 2017Co-Authors: Natalie Andrews, Laurence Bugeon, James Mcginty, Margaret J Dallman, Marie-christine Ramel, Sunil Kumar, Samuel P X Davis, Carys Hay, P M W FrenchAbstract:Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime Optical Projection Tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Forster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the Optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back Projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.
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OPTiM: Optical Projection Tomography integrated microscope using open-source hardware and software - Fig 4
2017Co-Authors: Thomas Watson, Laurence Bugeon, Natalie Andrews, Samuel Davis, Margaret D. Dallman, James McgintyAbstract:(a) External relay added to the camera port of the microscope for higher magnification standard OPT and remote focal scanning OPT (RFS). A continuation of the components within the microscope frame (Fig 1(C)), CM removable cube mirror. External RFS-OPT relay (red box): L3 achromatic doublet, M2/M3 mirror cubes, ETL electrically tunable lens. Conventional OPT relay (blue box): M4 mirror, L4/L5 achromatic doublets, AS variable iris acting as aperture stop. (b) The variable iris or ETL act as the aperture stop in the conventional or RFS OPT systems respectively. The size of the iris or scan range of the tunable lens determine the respective DoF, but axial displacement away from the conjugate pupil plane can lead to non-telecentric performance. The 3D axial PSF is no longer shift invariant, resulting in a depth dependant magnification. Note the point spread functions are scaled for illustrative purposes. (c,d) Simulation showing the static axial PSF (exterior panels) and effective axial PSF(central panels) for RFS-OPT and region of interest (RoI) OPT. (c) RFS-OPT with a scan range covering the full axial extent (d) RoI-OPT uses a smaller SR to increase the contrast to noise ratio over a desired region of interest. The RoI is tracked in depth, by adjusting the focal offset required to track the RoI during an acquisition (i.e as the sample rotates to each Projections angle).
Sunil Kumar - One of the best experts on this subject based on the ideXlab platform.
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single shot Optical Projection Tomography for high speed volumetric imaging
bioRxiv, 2021Co-Authors: Connor Darling, James Mcginty, Sunil Kumar, Samuel P X Davis, P M W FrenchAbstract:We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of Projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 Projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under {pound}5,000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.
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convolutional neural networks for reconstruction of undersampled Optical Projection Tomography data applied to in vivo imaging of zebrafish
Journal of Biophotonics, 2019Co-Authors: Samuel P X Davis, Yuriy Alexandrov, Sunil Kumar, Ajay Bhargava, Gabriela Da Silva Xavier, Guy A Rutter, Paul FrankelAbstract:Optical Projection Tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of Projection angles using the Radon transform, as is typically implemented with Optically cleared samples of the mm-to-cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts - reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to CS with 40% fewer Projections. We further show that diverse training data classes, for example ex vivo mouse tissue data, can be used for CNN-based reconstructions of OPT data of other species including live zebrafish. This article is protected by copyright. All rights reserved.
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exploiting patterned illumination and detection in Optical Projection Tomography conference presentation
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 2019Co-Authors: Samuel P X Davis, Teresa Correia, Laurence Bugeon, Paul Frankel, Marie-christine Ramel, Sunil Kumar, Laura Wisniewski, Simon R Arridge, Maggie Dallman, James McgintyAbstract:Optical Projection Tomography (OPT), the Optical equivalent of x-ray computed Tomography, reconstructs the 3D structure of a sample from a series of wide-field 2D Projections acquired at different angles [1]. OPT is used to map the Optical attenuation and/or fluorescence distributions of intact transparent samples without the need for mechanical sectioning. While it is typically applied to chemically cleared samples, it can also be used to image inherently transparent or weakly scattering live organisms including adult zebrafish up to ~1cm in diameter [2]. When applying OPT to live samples it is important to minimise the data acquisition time while maximising the image quality in the presence of scattering. The former issue can be addressed using compressive sensing to reduce the number of Projections required [3]. Scattered light can be rejected using structured illumination [4], but this removes emission from regions the excitation modulation does not reach and reduces the available dynamic range. To address this, we have explored the rejection of scattered light by acquiring Projections with parallel semi-confocal line illumination and detection in an approach we describe as slice-OPT (sl-OPT). The impact of Optical scattering can also be reduced by imaging at longer wavelengths [5]. We are exploring OPT in the NIR 1&2 spectral windows. However, exotic array detectors, e.g. for short wave infrared light, are costly and so we are also developing a single pixel camera [6] approach. We will present our progress applying these techniques to 3D imaging of vasculature and tumour burden in live adult zebrafish. [1] Sharpe et al, Science, vol. 296, Issue 5567, pp. 541-545, 2002. [2] Kumar et al, Oncotarget, vol. 7, no.28, pp. 43939-43948, 2016. [3] Correia et al, PloS one, vol. 10, no. 8, p. e0136213, 2015. [4] Kristensson et al, Optics express, vol. 20, no. 13, pp. 14437-14450, 2012. [5] Shi et al., Journal of Biophotonics, vol. 9, no. 1-2, pp. 38-43, 2016. [6] Duarte et al., IEEE Signal Processing Magazine, vol. 25, no. 2, pp. 83-91, 2008.
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slice illuminated Optical Projection Tomography
Optics Letters, 2018Co-Authors: Samuel P X Davis, Teresa Correia, Paul Frankel, James Mcginty, Sunil Kumar, Laura Wisniewski, Simon R Arridge, Paul M. W. FrenchAbstract:To improve the imaging performance of Optical Projection Tomography (OPT) in live samples, we have explored a parallelized implementation of semi-confocal line illumination and detection to discriminate against scattered photons. Slice-illuminated OPT (sl-OPT) improves reconstruction quality in scattering samples by reducing interpixel crosstalk at the cost of increased acquisition time. For in vivo imaging, this can be ameliorated through the use of compressed sensing on angularly undersampled OPT data sets. Here, we demonstrate sl-OPT applied to 3D imaging of bead phantoms and live adult zebrafish.
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functional imaging of live zebrafish using fluorescence lifetime Optical Projection Tomography conference presentation
Proceedings of SPIE, 2017Co-Authors: Natalie Andrews, Laurence Bugeon, James Mcginty, Margaret J Dallman, Marie-christine Ramel, Sunil Kumar, Samuel P X Davis, Carys Hay, P M W FrenchAbstract:Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime Optical Projection Tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Forster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the Optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back Projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.
Lingling Chen - One of the best experts on this subject based on the ideXlab platform.
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comparison of Optical Projection Tomography and light sheet fluorescence microscopy
Journal of Microscopy, 2019Co-Authors: Lingling Chen, An Liu, W Xiao, Liwei LiuAbstract:We present a numerical analysis and experimental characterisation of spatial resolution in Optical Projection Tomography (OPT) and light-sheet fluorescence microscopy (LSFM) using their 'standard' systems. Although both techniques provide spatial resolution at the micrometre scale for mesoscopic (millimetre to centimetre) samples, LSFM provides higher lateral (∼3 μm, ∼34% of OPT) but lower axial (∼25.8 μm, 295% of OPT) resolution as compared to OPT (∼8.75 μm, 100%) when imaging the same sample (∼2 mm). Moreover, OPT provides isotropic spatial resolution due to its rotational scanning which may reduce the ambiguity in 3D analysis, so it is more practically appropriate for relatively large samples. We also demonstrate the application performances of both techniques by imaging various biological tissues, illustrating their imaging ability at different spatial scales. LAY DESCRIPTION: Optical Projection Tomography (OPT) and light-sheet fluorescence microscopy (LSFM) are generally used to extract 3D information from relatively large biological tissues/organs/embryos or even some small animals. Both techniques have made a great progress in recent decades and have been widely applied in life science, medical research and so on. The different implementation features of these two techniques results in isotropic and anisotropic spatial resolution respectively, making a dilemma for the researchers to choose the appropriate system when imaging the samples with different size. So far, there is no study to numerically discuss the differences between their image formation properties and to adequately quantify their own strengths and limitations. In our work, we quantified the imaging behaviour in 'standard' OPT and LSFM using both numerical analysis and experimental characterisations, showing the relationship between spatial resolution and sample size in each system. We also demonstrated the detailed structure differences when imaging various biological tissues. We believe this work will be useful and can provide a reference for the 3D fluorescence-imaging-based researchers.
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mesoscopic in vivo 3 d tracking of sparse cell populations using angular multiplexed Optical Projection Tomography
Biomedical Optics Express, 2015Co-Authors: Lingling Chen, Natalie Andrews, Margaret J Dallman, Yuriy Alexandrov, Sunil Kumar, P M W French, James McgintyAbstract:We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and Optical Projection Tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different Projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 Projection angles. We demonstrate the application of this technique to mapping the 3-D neutrophil migration pattern observed over ~25.5 minutes in a live 2 day post-fertilisation transgenic LysC:GFP zebrafish embryo following a tail wound.
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Remote focal scanning Optical Projection Tomography with an electrically tunable lens
Biomedical Optics Express, 2014Co-Authors: Lingling Chen, Natalie Andrews, Douglas Kelly, Margaret J Dallman, Paul M. W. French, Sunil Kumar, James McgintyAbstract:We describe a remote focal scanning technique for Optical Projection Tomography (OPT) implemented with an electrically tunable lens (ETL) that removes the need to scan the specimen or objective lens. Using a 4× objective lens the average spatial resolution is improved by ∼46% and the light collection efficiency by a factor of ∼6.76, thereby enabling increased acquisition speed and reduced light dose. This convenient implementation is particularly appropriate for lower magnifications and larger sample diameters where axial objective scanning would encounter problems with speed and stability.
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simultaneous angular multiplexing Optical Projection Tomography at shifted focal planes
Optics Letters, 2013Co-Authors: Lingling Chen, Natalie Andrews, Paul Frankel, James Mcginty, Sunil Kumar, P M W FrenchAbstract:We describe an angular multiplexing technique for Optical Projection Tomography that improves resolution, signal-to-noise ratio, and imaging speed by ameliorating the trade-off between spatial resolution and depth of field and improving the light collection efficiency. Here we demonstrate that imaging at two orthogonal angular Projections simultaneously, focused on shifted planes in the sample, improves the average spatial resolution by ∼20% and the light collection efficiency by a factor of ∼4, thereby enabling increased acquisition speed and reduced light dose.
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incorporation of an experimentally determined mtf for spatial frequency filtering and deconvolution during Optical Projection Tomography reconstruction
Optics Express, 2012Co-Authors: Lingling Chen, Laurence Bugeon, Harriet B. Taylor, James Mcginty, Margaret J Dallman, Jonathan R Lamb, P M W FrenchAbstract:We demonstrate two techniques to improve the quality of reconstructed Optical Projection Tomography (OPT) images using the modulation transfer function (MTF) as a function of defocus experimentally determined from tilted knife-edge measurements. The first employs a 2-D binary filter based on the MTF frequency cut-off as an additional filter during back-Projection reconstruction that restricts the high frequency information to the region around the focal plane and progressively decreases the spatial frequency bandwidth with defocus. This helps to suppress “streak” artifacts in OPT data acquired at reduced angular sampling, thereby facilitating faster OPT acquisitions. This method is shown to reduce the average background by approximately 72% for an NA of 0.09 and by approximately 38% for an NA of 0.07 compared to standard filtered back-Projection. As a biological illustration, a Fli:GFP transgenic zebrafish embryo (3 days post-fertilisation) was imaged to demonstrate the improved imaging speed (a quarter of the acquisition time). The second method uses the MTF to produce an appropriate deconvolution filter that can be used to correct for the spatial frequency modulation applied by the imaging system.
Natalie Andrews - One of the best experts on this subject based on the ideXlab platform.
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functional imaging of live zebrafish using fluorescence lifetime Optical Projection Tomography conference presentation
Proceedings of SPIE, 2017Co-Authors: Natalie Andrews, Laurence Bugeon, James Mcginty, Margaret J Dallman, Marie-christine Ramel, Sunil Kumar, Samuel P X Davis, Carys Hay, P M W FrenchAbstract:Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime Optical Projection Tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Forster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the Optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back Projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.
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OPTiM: Optical Projection Tomography integrated microscope using open-source hardware and software - Fig 4
2017Co-Authors: Thomas Watson, Laurence Bugeon, Natalie Andrews, Samuel Davis, Margaret D. Dallman, James McgintyAbstract:(a) External relay added to the camera port of the microscope for higher magnification standard OPT and remote focal scanning OPT (RFS). A continuation of the components within the microscope frame (Fig 1(C)), CM removable cube mirror. External RFS-OPT relay (red box): L3 achromatic doublet, M2/M3 mirror cubes, ETL electrically tunable lens. Conventional OPT relay (blue box): M4 mirror, L4/L5 achromatic doublets, AS variable iris acting as aperture stop. (b) The variable iris or ETL act as the aperture stop in the conventional or RFS OPT systems respectively. The size of the iris or scan range of the tunable lens determine the respective DoF, but axial displacement away from the conjugate pupil plane can lead to non-telecentric performance. The 3D axial PSF is no longer shift invariant, resulting in a depth dependant magnification. Note the point spread functions are scaled for illustrative purposes. (c,d) Simulation showing the static axial PSF (exterior panels) and effective axial PSF(central panels) for RFS-OPT and region of interest (RoI) OPT. (c) RFS-OPT with a scan range covering the full axial extent (d) RoI-OPT uses a smaller SR to increase the contrast to noise ratio over a desired region of interest. The RoI is tracked in depth, by adjusting the focal offset required to track the RoI during an acquisition (i.e as the sample rotates to each Projections angle).
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remote focal scanning and sub volume Optical Projection Tomography
Cancer, 2016Co-Authors: Thomas Watson, Laurence Bugeon, Natalie Andrews, Margaret J Dallman, E Harry, P M W French, James McgintyAbstract:We present sub-volume Optical Projection Tomography utilising an electrically tunable lens and tracking technology. Applied to 3D fluorescent bead phantoms and zebrafish embryos, we demonstrate an improvement in resolution over conventional OPT.
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fluorescence lifetime Optical Projection Tomography and fret applied to visualizing apoptosis in live zebrafish larvae
Biomedical optics, 2016Co-Authors: Natalie Andrews, Nicola Lockwood, Louise Kerry, Antonina Frolov, Marie-christine Ramel, Douglas J. Kelly, Sean C. Warren, Yuriy Alexandrov, Sunil Kumar, Paul FrankelAbstract:We present the application of FLIM-OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed cleavable FRET biosensor for Caspase-3 as an indicator of gamma radiation induced apoptosis.
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Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with Optical Projection Tomography to map FRET biosensor activity in space and time
Journal of Biophotonics, 2016Co-Authors: Natalie Andrews, Nicola Lockwood, Louise Kerry, Antonina Frolov, Marie-christine Ramel, Douglas J. Kelly, Sean C. Warren, Yuriy Alexandrov, Sunil Kumar, Paul FrankelAbstract:Fluorescence lifetime imaging (FLIM) combined with Optical Projection Tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.
Jorge Ripoll - One of the best experts on this subject based on the ideXlab platform.
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Optical Projection Tomography via phase retrieval algorithms
Methods, 2017Co-Authors: Daniele Ancora, Jorge Ripoll, Diego Di Battista, Georgia Giasafaki, Stylianos Psycharakis, Evangelos Liapis, Giannis ZacharakisAbstract:We describe a computational method for accurate, quantitative tomographic reconstructions in Optical Projection Tomography, based on phase retrieval algorithms. Our method overcomes limitations imposed by light scattering in opaque tissue samples under the memory effect regime, as well as reduces artifacts due to mechanical movements, misalignments or vibrations. We make use of Gerchberg-Saxton algorithms, calculating first the autocorrelation of the object and then retrieving the associated phase under four numerically simulated measurement conditions. By approaching the task in such a way, we avoid the Projection alignment procedure, exploiting the fact that the autocorrelation sinogram is always aligned and centered. We thus propose two new, Projection-based, tomographic imaging flowcharts that allow registration-free imaging of opaque biological specimens and unlock three-dimensional tomographic imaging of hidden objects. Two main reconstruction approaches are discussed in the text, focusing on their efficiency in the tomographic retrieval and discussing their applicability under four different numerical experiments.
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polarization sensitive Optical Projection Tomography for muscle fiber imaging
Scientific Reports, 2016Co-Authors: Mengjie Fang, Alicia Arranz, Di Dong, Jorge Ripoll, Chaoting Zeng, Xiao Liang, Xin Yang, Hui Hui, Jie TianAbstract:Optical Projection Tomography (OPT) is a tool used for three-dimensional imaging of millimeter-scale biological samples, with the advantage of exhibiting isotropic resolution typically in the micron range. OPT can be divided into two types: transmission OPT (tOPT) and emission OPT (eOPT). Compared with eOPT, tOPT discriminates different tissues based on their absorption coefficient, either intrinsic or after specific staining. However, it fails to distinguish muscle fibers whose absorption coefficients are similar to surrounding tissues. To circumvent this problem, in this article we demonstrate a polarization sensitive OPT system which improves the detection and 3D imaging of muscle fibers by using polarized light. We also developed image acquisition and processing protocols that, together with the system, enable the clear visualization of muscles. Experimental results show that the muscle fibers of diaphragm and stomach, difficult to be distinguished in regular tOPT, were clearly displayed in our system, proving its potential use. Moreover, polarization sensitive OPT was fused with tOPT to investigate the stomach tissue comprehensively. Future applications of polarization sensitive OPT could be imaging other fiber-like structures such as myocardium or other tissues presenting high Optical anisotropy.
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in vivo Optical Tomography of small scattering specimens time lapse 3d imaging of the head eversion process in drosophila melanogaster
Scientific Reports, 2015Co-Authors: Alicia Arranz, Charalambos Savakis, Di Dong, Jorge Ripoll, Jie TianAbstract:Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current Optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window - thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously.
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helical Optical Projection Tomography
Optics Express, 2013Co-Authors: Alicia Arranz, Di Dong, Jie Tian, Shouping Zhu, Markus Rudin, Christos Tsatsanis, Jorge RipollAbstract:A new technique termed Helical Optical Projection Tomography (hOPT) has been developed with the aim to overcome some of the limitations of current 3D Optical imaging techniques. hOPT is based on Optical Projection Tomography (OPT) with the major difference that there is a translation of the sample in the vertical direction during the image acquisition process, requiring a new approach to image reconstruction. Contrary to OPT, hOPT makes possible to obtain 3D-Optical images of intact long samples without imposing limits on the sample length. This has been tested using hOPT to image long murine tissue samples such as spinal cords and large intestines. Moreover, 3D-reconstructed images of the colon of DSS-treated mice, a model for Inflammatory Bowel Disease, allowed the identification of the structural alterations. Finally, the geometry of the hOPT device facilitates the addition of a Selective Plane Illumination Microscopy (SPIM) arm, providing the possibility of delivering high resolution images of selected areas together with complete volumetric information.
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Automated Recovery of the Center of Rotation in Optical Projection Tomography in the Presence of Scattering
IEEE Journal of Biomedical and Health Informatics, 2013Co-Authors: Di Dong, Charalambos Savakis, Jie Tian, Jens V Stein, Shouping Zhu, Chenghu Qin, Varsha Kumar, Stephan Oehler, Jorge RipollAbstract:Finding the center of rotation is an essential step for accurate 3-D reconstruction in Optical Projection Tomography. Unfortunately, current methods are not convenient since they require either prior scanning of a reference phantom, small structures of high intensity existing in the specimen, or active participation during the centering procedure. To solve these problems this paper proposes a fast and automatic center of rotation search method making use of parallel programming in graphics processing units. Our method is based on a two step search approach making use only of those sections of the image with high signal-to-noise ratio. We have tested this method both in nonscattering ex vivo samples and in in vivo specimens with a considerable contribution of scattering such as Drosophila melanogaster pupae, recovering in all cases the center of rotation with a precision 1/4 pixel or less.