Biological and Medical Physics, Biomedical Engineering, 2019
In this chapter, we present a summary of research that uses silicon to enhance the imaging resolu... more In this chapter, we present a summary of research that uses silicon to enhance the imaging resolution and to push it toward the region of nanoscopy. The silicon has a nonlinear effect called the plasma dispersion effect (PDE), which can be used instead of the fluorescent dye in order to realize a stimulated emission depletion (STED) like microscopic imaging configuration. The silicon can be encapsulated into nanoparticles while the encapsulation may be biocompatible and yield the first step toward label-free bioimaging. The encapsulation may even be metallic to enhance the obtainable effect and to yield resolution enhancement at lower requirements from the pump beam. The silicon can even be used directly as a wafer but then the super-resolving imaging is mainly aimed at failure analysis of micro-electronic circuitry. When the silicon is encapsulated with metallic layer or when it is in the form of a wafer, while being illuminated with a pump beam (in visible wavelength being absorbed by the silicon) it modifies the imaging point spread function (PSF) of the probe beam (a near infra-red wavelength) into a doughnut-like shape, through the utilization of the PDE. This modified PSF has spatial components smaller than the diffraction limit, and by scanning the sample with it, super resolution can be achieved.
2017 IEEE International Ultrasonics Symposium (IUS), 2017
The recent development of ultrafast ultrasound localization microscopy (Errico et al., Nature, 20... more The recent development of ultrafast ultrasound localization microscopy (Errico et al., Nature, 2015) provides new opportunities for imaging the vasculature with sub-diffraction resolution. However, the large number of images to be recorded requires acquisitions lasting several minutes. Therefore, physiological motion far larger than the achievable resolution presents challenges for moving tissues. In this work, single microbubbles (MB) were localized in vivo in a rat kidney using a dedicated imaging sequence allowing MB tracking and motion correction. Blood velocity in small vessels (<2 mm/s) was estimated demonstrating the potential of this technique to improve vascular characterization.
Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with tra... more Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. ...
Ultrasound images are severely degraded by the presence of obstacles such as bones and air gaps a... more Ultrasound images are severely degraded by the presence of obstacles such as bones and air gaps along the beam path. This paper describes a method for imaging structures that are distal to obstacles that are otherwise impenetrable to ultrasound. The method uses an optically-inspired holographic algorithm to beam-shape the emitted ultrasound field in order to bypass the obstacle and place the beam focus beyond the obstruction. The resulting performance depends on the transducer aperture, the size and position of the obstacle, and the position of the target. Improvement compared to standard ultrasound imaging is significant for obstacles for which the width is larger than one fourth of the transducer aperture and the depth is within a few centimeters of the transducer. For such cases, the improvement in focal intensity at the location of the target reaches 30-fold, and the improvement in peak-to-side-lobe ratio reaches 3-fold. The method can be implemented in conventional ultrasound s...
Structured illumination microscopy is an optical method to increase the spatial resolution of wid... more Structured illumination microscopy is an optical method to increase the spatial resolution of wide-field fluorescence imaging beyond the diffraction limit by applying a spatially structured illumination light. Here, we extend this concept to facilitate super-resolution ultrasound imaging by manipulating the transmitted sound field to encode the high spatial frequencies into the observed image through aliasing. Post processing is applied to precisely shift the spectral components to their proper positions in -space and effectively double the spatial resolution of the reconstructed image compared to one-way focusing. The method has broad application, including the detection of small lesions for early cancer diagnosis, improving the detection of the borders of organs and tumors, and enhancing visualization of vascular features. The method can be implemented with conventional ultrasound systems, without the need for additional components. The resulting image enhancement is demonstrated ...
The recent development of ultrasound localization microscopy, where individual microbubbles (cont... more The recent development of ultrasound localization microscopy, where individual microbubbles (contrast agents) are detected and tracked within the vasculature, provides new opportunities for imaging the vasculature of entire organs with a spatial resolution below the diffraction limit. In stationary tissue, recent studies have demonstrated a theoretical resolution on the order of microns. In this work, single microbubbles were localized in vivo in a rat kidney using a dedicated high frame rate imaging sequence. Organ motion was tracked by assuming rigid motion (translation and rotation) and appropriate correction was applied. In contrast to previous work, coherence-based non-linear phase inversion processing was used to reject tissue echoes while maintaining echoes from very slowly moving microbubbles. Blood velocity in the small vessels was estimated by tracking microbubbles, demonstrating the potential of this technique to improve vascular characterization. Previous optical studies...
Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biolog... more Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold nanoparticles tagged sample using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is then further enhanced also for tracking of single fluorescent particles within a three dimensional (3D) cellular environment based on image processing algorithms that can significantly increases localization accuracy of the 3D point spread function in respect to regular Gaussian fitting. All proposed concepts are validated both on simulated data as well as experimentally.
2016 15th Workshop on Information Optics (WIO), 2016
In our paper we will report the first step of a new method for changing the diffraction limited p... more In our paper we will report the first step of a new method for changing the diffraction limited point spread function through the utilization of the plasma dispersion effect of silicon coated gold nanoparticles to achieve sub wavelength resolution. The plasma dispersion effect can change the optical coefficients of the silicon, under illumination with a focused laser beam. This laser illumination has a Gaussian profile with a higher intensity around its peak. As a result, the plasma dispersion effect is more pronounces at the center of the illumination. As a consequence, the reflected light from probe beam at the near infra-red region has a sub wavelength dip that overlaps with the location of the pump illumination peak. This dip has a higher spatial frequency than an ordinary Gaussian, which enables to achieve super resolution..
Super-resolution localization microscopy can overcome the diffraction limit and achieve a tens of... more Super-resolution localization microscopy can overcome the diffraction limit and achieve a tens of order improvement in resolution. It requires labeling the sample with fluorescent probes followed with their repeated cycles of activation and photobleaching. This work presents an alternative approach that is free from direct labeling and does not require the activation and photobleaching cycles. Fluorescently labeled gold nanoparticles in a solution are distributed on top of the sample. The nanoparticles move in a random Brownian motion, and interact with the sample. By obscuring different areas in the sample, the nanoparticles encode the sub-wavelength features. A sequence of images of the sample is captured and decoded by digital post processing to create the super-resolution image. The achievable resolution is limited by the additive noise and the size of the nanoparticles. Regular nanoparticles with diameter smaller than 100nm are barely seen in a conventional bright field microscope, thus fluorescently labeled gold nanoparticles were used, with proper
Abstract. This paper presents a method for modifying the point spread function (PSF) into a dough... more Abstract. This paper presents a method for modifying the point spread function (PSF) into a doughnut-like shape, through the utilization of the plasma dispersion effect (PDE) of silicon-coated gold nanoparticles. This modified PSF has spatial components smaller than the diffraction limit, and by scanning the sample with it, super-resolution can be achieved. The sample is illuminated using two laser beams. The first is the pump, with a wavelength in the visible region that creates a change in the refractive index of the silicon coating due to the PDE. This creates a change in the localized surface plasmon resonance wavelength. Since the pump beam has a Gaussian profile, the high intensity areas of the beam experience the highest refractive index change. When the second beam (i.e., the probe) illuminates the sample with a near-infrared wavelength, this change in the refractive index is transformed into a change in the PSF profile. The ordinary Gaussian shape is transformed into a doughnut shape, with higher spatial frequencies, which enables one to achieve super-resolution by scanning the specimen using this PSF. This is a step toward the creation of a nonfluorescent nanoscope.
Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biolog... more Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold-particle tagged samples using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is presented and validated both on simulated data as well as experimentally.
Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology, May 15, 2015
The temporal flickering of contrast agents that labels a biological sample is a unique modality f... more The temporal flickering of contrast agents that labels a biological sample is a unique modality for cellular imaging with single molecule sensitivity. It improves the signal-to-noise ratio statistics associated with the noisy in vivo environment and has promising applications in single particle tracking and super-resolution microscopy techniques. The flickering can be triggered either statistically through the mechanism of temporal fluctuations of the emitter or through external modulation. The enriching toolbox of contrast agents that are feasible for biomedical imaging for the flickering methods will be discussed, with emphasis on the emerging field of flickering gold nanoparticles and the lock-in detection mechanism. For further resources related to this article, please visit the WIREs website.
Localization microscopy provides valuable insights into cellular structures and is a rapidly deve... more Localization microscopy provides valuable insights into cellular structures and is a rapidly developing field. The precision is mainly limited by additive noise and the requirement for single molecule imaging that dictates a low density of activated emitters in the field of view. In this paper we present a technique aimed for noise reduction and improved localization accuracy. The method has two steps; the first is the imaging of gold nanoparticles that labels targets of interest inside biological cells using a lock-in technique that enables the separation of the signal from the wide spread spectral noise. The second step is the application of the K-factor nonlinear image decomposition algorithm on the obtained image, which improves the localization accuracy that can reach 5nm and enables the localization of overlapping particles at minimal distances that are closer by 65% than conventional methods.
In this paper we present a technique aimed for simultaneous detection of multiple types of gold n... more In this paper we present a technique aimed for simultaneous detection of multiple types of gold nanoparticles (GNPs) within a biological sample, using lock-in detection. We image the sample using a number of modulated laser beams that correspond to the number of GNP species that label a given sample. The final image where the GNPs are spatially separated is obtained computationally. The proposed method enables the simultaneous superresolved imaging of different areas of interest within biological sample and also the spatial separation of GNPs at sub-diffraction distances, making it a useful tool in the study of intracellular trafficking pathways in living cells.
The use of a two-dimensional Barker-based array in the conventional time multiplexing super-resol... more The use of a two-dimensional Barker-based array in the conventional time multiplexing super-resolution (TMSR) technique was recently presented [Opt. Lett.40, 163-165 (2015)OPLEDP0146-959210.1364/OL.40.000163]. It enables achieving a two-dimensional SR image using only a one-dimensional scan, by exploiting its unique auto-correlation property. In this Letter, we refine the method using a mismatched array for the decoding process. The cross-correlation between the Barker-based array and the mismatched array has a perfect peak-to-sidelobes ratio, making it ideal for the SR process. Also, we propose the projection of this array onto the object using a phase-only spatial light modulator. Projecting the array eliminates the need for printing it, mechanically shifting it, and having a direct contact with the object, which is not feasible in many imaging applications. 13 phase masks, which generate shifted Barker-based arrays, were designed using a revised Gerchberg-Saxton algorithm. A sequ...
Biological and Medical Physics, Biomedical Engineering, 2019
In this chapter, we present a summary of research that uses silicon to enhance the imaging resolu... more In this chapter, we present a summary of research that uses silicon to enhance the imaging resolution and to push it toward the region of nanoscopy. The silicon has a nonlinear effect called the plasma dispersion effect (PDE), which can be used instead of the fluorescent dye in order to realize a stimulated emission depletion (STED) like microscopic imaging configuration. The silicon can be encapsulated into nanoparticles while the encapsulation may be biocompatible and yield the first step toward label-free bioimaging. The encapsulation may even be metallic to enhance the obtainable effect and to yield resolution enhancement at lower requirements from the pump beam. The silicon can even be used directly as a wafer but then the super-resolving imaging is mainly aimed at failure analysis of micro-electronic circuitry. When the silicon is encapsulated with metallic layer or when it is in the form of a wafer, while being illuminated with a pump beam (in visible wavelength being absorbed by the silicon) it modifies the imaging point spread function (PSF) of the probe beam (a near infra-red wavelength) into a doughnut-like shape, through the utilization of the PDE. This modified PSF has spatial components smaller than the diffraction limit, and by scanning the sample with it, super resolution can be achieved.
2017 IEEE International Ultrasonics Symposium (IUS), 2017
The recent development of ultrafast ultrasound localization microscopy (Errico et al., Nature, 20... more The recent development of ultrafast ultrasound localization microscopy (Errico et al., Nature, 2015) provides new opportunities for imaging the vasculature with sub-diffraction resolution. However, the large number of images to be recorded requires acquisitions lasting several minutes. Therefore, physiological motion far larger than the achievable resolution presents challenges for moving tissues. In this work, single microbubbles (MB) were localized in vivo in a rat kidney using a dedicated imaging sequence allowing MB tracking and motion correction. Blood velocity in small vessels (<2 mm/s) was estimated demonstrating the potential of this technique to improve vascular characterization.
Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with tra... more Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. ...
Ultrasound images are severely degraded by the presence of obstacles such as bones and air gaps a... more Ultrasound images are severely degraded by the presence of obstacles such as bones and air gaps along the beam path. This paper describes a method for imaging structures that are distal to obstacles that are otherwise impenetrable to ultrasound. The method uses an optically-inspired holographic algorithm to beam-shape the emitted ultrasound field in order to bypass the obstacle and place the beam focus beyond the obstruction. The resulting performance depends on the transducer aperture, the size and position of the obstacle, and the position of the target. Improvement compared to standard ultrasound imaging is significant for obstacles for which the width is larger than one fourth of the transducer aperture and the depth is within a few centimeters of the transducer. For such cases, the improvement in focal intensity at the location of the target reaches 30-fold, and the improvement in peak-to-side-lobe ratio reaches 3-fold. The method can be implemented in conventional ultrasound s...
Structured illumination microscopy is an optical method to increase the spatial resolution of wid... more Structured illumination microscopy is an optical method to increase the spatial resolution of wide-field fluorescence imaging beyond the diffraction limit by applying a spatially structured illumination light. Here, we extend this concept to facilitate super-resolution ultrasound imaging by manipulating the transmitted sound field to encode the high spatial frequencies into the observed image through aliasing. Post processing is applied to precisely shift the spectral components to their proper positions in -space and effectively double the spatial resolution of the reconstructed image compared to one-way focusing. The method has broad application, including the detection of small lesions for early cancer diagnosis, improving the detection of the borders of organs and tumors, and enhancing visualization of vascular features. The method can be implemented with conventional ultrasound systems, without the need for additional components. The resulting image enhancement is demonstrated ...
The recent development of ultrasound localization microscopy, where individual microbubbles (cont... more The recent development of ultrasound localization microscopy, where individual microbubbles (contrast agents) are detected and tracked within the vasculature, provides new opportunities for imaging the vasculature of entire organs with a spatial resolution below the diffraction limit. In stationary tissue, recent studies have demonstrated a theoretical resolution on the order of microns. In this work, single microbubbles were localized in vivo in a rat kidney using a dedicated high frame rate imaging sequence. Organ motion was tracked by assuming rigid motion (translation and rotation) and appropriate correction was applied. In contrast to previous work, coherence-based non-linear phase inversion processing was used to reject tissue echoes while maintaining echoes from very slowly moving microbubbles. Blood velocity in the small vessels was estimated by tracking microbubbles, demonstrating the potential of this technique to improve vascular characterization. Previous optical studies...
Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biolog... more Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold nanoparticles tagged sample using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is then further enhanced also for tracking of single fluorescent particles within a three dimensional (3D) cellular environment based on image processing algorithms that can significantly increases localization accuracy of the 3D point spread function in respect to regular Gaussian fitting. All proposed concepts are validated both on simulated data as well as experimentally.
2016 15th Workshop on Information Optics (WIO), 2016
In our paper we will report the first step of a new method for changing the diffraction limited p... more In our paper we will report the first step of a new method for changing the diffraction limited point spread function through the utilization of the plasma dispersion effect of silicon coated gold nanoparticles to achieve sub wavelength resolution. The plasma dispersion effect can change the optical coefficients of the silicon, under illumination with a focused laser beam. This laser illumination has a Gaussian profile with a higher intensity around its peak. As a result, the plasma dispersion effect is more pronounces at the center of the illumination. As a consequence, the reflected light from probe beam at the near infra-red region has a sub wavelength dip that overlaps with the location of the pump illumination peak. This dip has a higher spatial frequency than an ordinary Gaussian, which enables to achieve super resolution..
Super-resolution localization microscopy can overcome the diffraction limit and achieve a tens of... more Super-resolution localization microscopy can overcome the diffraction limit and achieve a tens of order improvement in resolution. It requires labeling the sample with fluorescent probes followed with their repeated cycles of activation and photobleaching. This work presents an alternative approach that is free from direct labeling and does not require the activation and photobleaching cycles. Fluorescently labeled gold nanoparticles in a solution are distributed on top of the sample. The nanoparticles move in a random Brownian motion, and interact with the sample. By obscuring different areas in the sample, the nanoparticles encode the sub-wavelength features. A sequence of images of the sample is captured and decoded by digital post processing to create the super-resolution image. The achievable resolution is limited by the additive noise and the size of the nanoparticles. Regular nanoparticles with diameter smaller than 100nm are barely seen in a conventional bright field microscope, thus fluorescently labeled gold nanoparticles were used, with proper
Abstract. This paper presents a method for modifying the point spread function (PSF) into a dough... more Abstract. This paper presents a method for modifying the point spread function (PSF) into a doughnut-like shape, through the utilization of the plasma dispersion effect (PDE) of silicon-coated gold nanoparticles. This modified PSF has spatial components smaller than the diffraction limit, and by scanning the sample with it, super-resolution can be achieved. The sample is illuminated using two laser beams. The first is the pump, with a wavelength in the visible region that creates a change in the refractive index of the silicon coating due to the PDE. This creates a change in the localized surface plasmon resonance wavelength. Since the pump beam has a Gaussian profile, the high intensity areas of the beam experience the highest refractive index change. When the second beam (i.e., the probe) illuminates the sample with a near-infrared wavelength, this change in the refractive index is transformed into a change in the PSF profile. The ordinary Gaussian shape is transformed into a doughnut shape, with higher spatial frequencies, which enables one to achieve super-resolution by scanning the specimen using this PSF. This is a step toward the creation of a nonfluorescent nanoscope.
Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biolog... more Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold-particle tagged samples using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is presented and validated both on simulated data as well as experimentally.
Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology, May 15, 2015
The temporal flickering of contrast agents that labels a biological sample is a unique modality f... more The temporal flickering of contrast agents that labels a biological sample is a unique modality for cellular imaging with single molecule sensitivity. It improves the signal-to-noise ratio statistics associated with the noisy in vivo environment and has promising applications in single particle tracking and super-resolution microscopy techniques. The flickering can be triggered either statistically through the mechanism of temporal fluctuations of the emitter or through external modulation. The enriching toolbox of contrast agents that are feasible for biomedical imaging for the flickering methods will be discussed, with emphasis on the emerging field of flickering gold nanoparticles and the lock-in detection mechanism. For further resources related to this article, please visit the WIREs website.
Localization microscopy provides valuable insights into cellular structures and is a rapidly deve... more Localization microscopy provides valuable insights into cellular structures and is a rapidly developing field. The precision is mainly limited by additive noise and the requirement for single molecule imaging that dictates a low density of activated emitters in the field of view. In this paper we present a technique aimed for noise reduction and improved localization accuracy. The method has two steps; the first is the imaging of gold nanoparticles that labels targets of interest inside biological cells using a lock-in technique that enables the separation of the signal from the wide spread spectral noise. The second step is the application of the K-factor nonlinear image decomposition algorithm on the obtained image, which improves the localization accuracy that can reach 5nm and enables the localization of overlapping particles at minimal distances that are closer by 65% than conventional methods.
In this paper we present a technique aimed for simultaneous detection of multiple types of gold n... more In this paper we present a technique aimed for simultaneous detection of multiple types of gold nanoparticles (GNPs) within a biological sample, using lock-in detection. We image the sample using a number of modulated laser beams that correspond to the number of GNP species that label a given sample. The final image where the GNPs are spatially separated is obtained computationally. The proposed method enables the simultaneous superresolved imaging of different areas of interest within biological sample and also the spatial separation of GNPs at sub-diffraction distances, making it a useful tool in the study of intracellular trafficking pathways in living cells.
The use of a two-dimensional Barker-based array in the conventional time multiplexing super-resol... more The use of a two-dimensional Barker-based array in the conventional time multiplexing super-resolution (TMSR) technique was recently presented [Opt. Lett.40, 163-165 (2015)OPLEDP0146-959210.1364/OL.40.000163]. It enables achieving a two-dimensional SR image using only a one-dimensional scan, by exploiting its unique auto-correlation property. In this Letter, we refine the method using a mismatched array for the decoding process. The cross-correlation between the Barker-based array and the mismatched array has a perfect peak-to-sidelobes ratio, making it ideal for the SR process. Also, we propose the projection of this array onto the object using a phase-only spatial light modulator. Projecting the array eliminates the need for printing it, mechanically shifting it, and having a direct contact with the object, which is not feasible in many imaging applications. 13 phase masks, which generate shifted Barker-based arrays, were designed using a revised Gerchberg-Saxton algorithm. A sequ...
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