Atomic Force Microscope (AFM) imaging, due to the scanning method of recording, requires significant recording
time for examination of wide sample area. In contrast, digital holographic microscopy (DHM), owing to the wide-field
method, allows recording of the hologram in very fast rate which could be numerically analyzed to reveal surface of the
sample with axial resolution at the nanometer scale. However, DHM yields quantitative phase properties of the sample,
and therefore sensitive to changes in refractive index along with physical thickness. Therefore, to accurately determine
the refractive index map, it is imperative to estimate the physical thickness map of the sample. This was achieved by
AFM imaging. Further, since the transverse resolution of DHM is limited by diffraction limit, co-registration of AFM
image provided higher transverse resolution at nanometer scale. The interference of the AFM probe was observed to be
minimal during simultaneous AFM and DHM recording due to the transparent nature and bent configuration of the
optical fiber based AFM cantilever. Integration of DHM and AFM led to realization of a powerful platform for
nanoscale imaging. The integrated AFM-DHM system was built on an inverted fluorescence microscope to enable
fluorescence imaging of the sample. The integrated system was employed to analyze fluorescent polystyrene
microspheres, two-photon polymerized microstructures and red blood cells.
Red blood cells (RBC) possess unique viscoelastic characteristics which allow them to pass through capillaries
narrower than their size. Measurement of viscoelastic property of cells (e.g. RBC) in low-force regime is of high
significance as it represents conditions of membrane fluctuation in response to physiological conditions.
Estimation of visco-elastic properties of RBC requires measurement of extent of deformation in RBC subjected
to known force. Optical tweezers, being gentle and absolutely sterile, are emerging as the tool of choice for
application of localized force on cells. However, stretching of RBC in very low force regime has not been
quantified. Further, though deformations in transverse directions have been measured, vertical deformations due
to stretching of cells cannot be quantified by classical microscopic images. Here, we report realization of offaxis
digital holographic microscopy (DHM) for highly sensitive axial changes in RBC shape due to stretching
by optical tweezers without attaching microscopic beads. The RBC was stretched in axial direction with
nanometer precision by change of divergence of the trapping beam. The obtained deformation patterns were
compared with the axial position of the tweezers focus. Since the pathophysiology of progression of diseases
like malaria and cancer is reflected in the biophysical (both mechanical and material) properties of the cells, it is
possible to identify the changes by simultaneous measurement of refractive index and elasticity using this
approach.
In this paper, we analyzed the retinal and choroidal blood vasculature in the posterior segment of the human eye with
optimized color Doppler and Doppler variance optical coherence tomography. Depth-resolved structure, color Doppler
and Doppler variance images were compared. Blood vessels down to capillary level were able to be obtained with the
optimized optical coherence color Doppler and Doppler variance method. For in-vivo imaging of human eyes, bulkmotion
induced bulk phase must be identified and removed before using color Doppler method. It was found that the
Doppler variance method is not sensitive to bulk motion and the method can be used without removing the bulk phase. A
novel, simple and fast segmentation algorithm to indentify retinal pigment epithelium (RPE) was proposed and used to
segment the retinal and choroidal layer. The algorithm was based on the detected OCT signal intensity difference
between different layers. A spectrometer-based Fourier domain OCT system with a central wavelength of 890 nm and
bandwidth of 150nm was used in this study. The 3-dimensional imaging volume contained 120 sequential two
dimensional images with 2048 A-lines per image. The total imaging time was 12 seconds and the imaging area was 5x5
mm2.
While optical tweezers have been widely used for the manipulation and organization of microscopic objects in three
dimensions, observing the manipulated objects along axial direction has been quite challenging. In order to visualize
organization and orientation of objects along axial direction, we report development of a Digital holographic microscopy
combined with optical tweezers. Digital holography is achieved by use of a modified Mach-Zehnder interferometer with
digital recording of interference pattern of the reference and sample laser beams by use of a single CCD camera. In this
method, quantitative phase information is retrieved dynamically with high temporal resolution, only limited by frame
rate of the CCD. Digital focusing, phase-unwrapping as well as online analysis and display of the quantitative phase
images was performed on a software developed on LabView platform. Since phase changes observed in DHOT is very
sensitive to optical thickness of trapped volume, estimation of number of particles trapped in the axial direction as well
as orientation of non-spherical objects could be achieved with high precision. Since in diseases such as malaria and
diabetics, change in refractive index of red blood cells occurs, this system can be employed to map such disease-specific
changes in biological samples upon immobilization with optical tweezers.
Interaction of red blood cells (RBC) with optical tweezers has been found to differ under varied physiological and
pathological conditions as compared to its normal conditions. Earlier, we reported difference in rotation of trapped RBC
in hypertonic conditions for detection of malaria infection. Disk-like RBC when trapped in optical tweezers get oriented
in the vertical plane to maximize interaction with trapping beam. However, classical bright field, phase contrast or epifluorescence
microscopy cannot confirm its orientation, thus leading to ambiguous conclusions such as folding of RBC
during trapping by some researchers. Now, with use of digital holographic microscopy (DHM), we achieved high axial
sensitivity that confirmed orientation of trapped red blood cell. Further, DHM enabled quantitative phase imaging of
RBC under hypertonic condition. Dynamic changes of rotating RBC under optical tweezers at different trapping laser
power were evaluated by the use of DHM. The deviation from linear dependence of rotation speed of RBC on laser
power, was attributed towards deformation of RBC shape due to higher laser power (or speed).
We report the use of spectral Doppler optical coherence tomography imaging (SDOCTI) for quantitative evaluation of dynamic blood circulation before and after a localized ischemic stroke in a mouse model. Rose Bengal photodynamic therapy (PDT) is used as a noninvasive means for inducing localized ischemia in cortical microvasculature of the mouse. Fast, repeated Doppler optical coherence tomography scans across vessels of interest are performed to record flow dynamic information with high temporal resolution. Doppler-angle-independent flow indices are used to quantify vascular conditions before and after the induced ischemia by the photocoagulation of PDT. The higher (or lower) flow resistive indices are associated with higher (or lower) resistance states that are confirmed by laser speckle flow index maps (of laser speckle imaging). Our in vivo experiments shows that SDOCTI can provide complementary quantified flow information that is an alternative to blood volume measurement, and can be used as a means for cortical microvasculature imaging well suited for small animal studies.
Optical methods have been widely used in basic neuroscience research to study the cerebral blood flow dynamics in order to overcome the low spatial resolution associated with magnetic resonance imaging and positron emission tomography. Although laser Doppler imaging and laser speckle imaging can map out en face cortical hemodynamics and
columns, depth resolution is not available. Two-photon microscopy has been used for mapping cortical activity. However, flow measurement requires fluorescent dye injection, which can be problematic. The noninvasive and high resolution tomographic capabilities of optical coherence tomography make it a promising technique for mapping depth resolved cortical blood flow. Here, we present a functional Doppler optical coherence tomography (OCT) imaging modality for quantitative evaluation of cortical blood flow in a mouse model. Fast, repeated, Doppler OCT scans across
a vessel of interest were performed to record flow dynamic information with a high temporal resolution of the cardiac
cycles. Spectral Doppler analysis of continuous Doppler images demonstrates how the velocity components and longitudinally projected flow-volume-rate change over time, thereby providing complementary temporal flow information to the spatially distributed flow information of Doppler OCT. The proposed functional Doppler OCT
imaging modality can be used to diagnose vessel stenosis/blockage or monitor blood flow changes due to pharmacological agents/neuronal activities. Non-invasive in-vivo mice experiments were performed to verify the capabilities of function Doppler OCT.
Electromechanical reshaping (EMR) of cartilage is a promising noninvasive technique with potential for broad
application in reconstructive surgery. EMR involves applying direct current electrical fields to localized stress regions
and initiating a series of oxidation-reduction reactions, thus effecting a shape change. Previous EMR studies have
focused on macroscopic structural measurements of the shape change effect or monitoring of electrical current flow.
Only limited investigation of structural changes in the tissue at the histologic level have been performed, and not in real
time. This study is the first to use optical coherence tomography (OCT) to examine structural changes in cartilage during
EMR. Two platinum needle electrodes were inserted into fixed rectangular rabbit nasal septal cartilage specimens. The
spectral domain OCT probe was then positioned above the section of cartilage in which the anode needle was inserted. A
constant voltage of 6V was applied for 3 minutes, and images were obtained (8 frames/second). OCT was also performed
in specimens undergoing dehydration under ambient conditions and during pH changes produced by the addition of HCl,
as both processes accompany EMR. The OCT data identified distinct findings among the three conditions, suggesting
that EMR causes a much greater degree of reshaping on a molecular level than dehydration or a change in pH alone.
OCT provides a means to gauge structural changes in the tissue matrix during EMR. The application of OCT to image
the EMR process will add to our understanding of the mechanisms of action involved and potentially facilitate
optimization of this process.
KEYWORDS: Optical coherence tomography, Signal to noise ratio, Calibration, Image filtering, Digital filtering, Tomography, Optical filters, Adaptive optics, Data acquisition, Data analysis
Empirical mode decomposition (EMD) is a new adaptive data analysis method in which the analyzed data is decomposed into a limited number of intrinsic mode functions (IMFs) through a sifting
process. One problem with EMD is mode mixing, which has been solved by Wu et al using ensemble EMD (EEMD). In this paper, we applied the EEMD method to data acquired from optical coherence
tomography (OCT) to improve the image quality. First, the original OCT fringe data is converted from linear wavelength to linear frequency through a calibration process. Second, the calibrated data is decomposed into different IMFs by EEMD. Third, the physical meaning of different IMFs was analyzed.
Fourth, IMFs that represented noise were removed from the calibrated fringe data. The noise removed
fringe data was then Fourier transformed to get depth information. EEMD was found to be able to separate
different frequency noise into different IMFs. The signal to noise ratio of OCT image was improved by
removing the IMFs that represent noise from the acquired fringe data.
KEYWORDS: Optical coherence tomography, Speckle, 3D image processing, Image processing, Signal attenuation, Signal processing, Signal to noise ratio, Interference (communication), Wavelets, Biomedical optics
Optical coherence tomography is an emerging non-invasive technology that provides high resolution, cross-sectional
tomographic images of internal structures of specimens. It holds great potentials for a wide variety of applications,
especially in the field of biomedical imaging. OCT images, however, are usually degraded by significant speckle noise.
Here we report a 3D approach to attenuating speckle noise in OCT images. This approach is based on the 3D curvelet
transform, and is conveniently controlled by a single parameter that determines the threshold in the curvelet domain.
Unlike 2D approaches which only consider information in individual images, 3D processing, by analyzing all images in
a volume simultaneously, has the advantage of also taking the information between images into account. This, coupled
with the curvelet transform's nearly optimal sparse representation of curved edges that are common in OCT images,
provides a simple yet powerful platform for speckle attenuation. We show the approach suppresses a significant amount
of speckle noise, and in the mean time preserves and thus reveals many subtle features that could get attenuated in other approaches.
KEYWORDS: Optical coherence tomography, Injuries, Image processing, 3D image processing, Microelectromechanical systems, Reconstruction algorithms, In vivo imaging, 3D modeling, 3D image reconstruction, Endoscopes
We report on the feasibility of rapid, high resolution, 3-dimensional swept source optical coherence tomography (3D
SSOCT) to detect early airway injury changes following smoke inhalation exposure in a rabbit model. The SSOCT
system obtains 3-D helical scanning using a microelectromechanical system (MEMS) motor based endoscope. Real-time
2-D data processing and image display at the speed of 20 frames per second are achieved by adopting the technique of
shared-memory parallel computing. Longitudinal images are reconstructed via an image processing algorithm to remove
motion artifacts caused by ventilation and pulse. We demonstrate the ability of the SSOCT system to detect increases in
tracheal and bronchial airway thickness that occurs shortly after smoke exposure.
We present a combined optical Doppler tomography/spectral Doppler imaging modality to quantitatively evaluate the
dynamic blood circulation and the artery blockage before and after a localized ischemic stroke in a mouse model. Optical
Doppler Tomography (ODT) combines the Doppler principle with optical coherence tomography for noninvasive
localization and measurement of particle flow velocity in highly scattering media with micrometer scale spatial
resolution. Spectral Doppler imaging (SDI) provides complementary temporal flow information to the spatially
distributed flow information of Doppler imaging. Fast, repeated, ODT scans across an entire vessel were performed to
record flow dynamic information with high temporal resolution of cardiac cycles. Spectral Doppler analysis of
continuous Doppler images demonstrates how the velocity components and longitudinally projected flow-volume-rate
change over time for scatters within the imaging volume using spectral Doppler waveforms. Furthermore, vascular
conditions can be quantified with various Doppler-angle-independent flow indices. Non-invasive in-vivo mice
experiments were performed to evaluate microvascular blood circulation of a localized ischemic stroke mouse model.
We demonstrate the use of Doppler variance (standard deviation) imaging for 3-D in vivo angiography in the human eye. In addition to the regular optical Doppler tomography velocity and structural images, we use the variance of blood flow velocity to map the retina and choroid vessels. Variance imaging is subject to bulk motion artifacts as in phase-resolved Doppler imaging, and a histogram-based method is proposed for bulk-motion correction in variance imaging. Experiments were performed to demonstrate the effectiveness of the proposed method for 3-D vasculature imaging of human retina and choroid.
We report on the feasibility of rapid, high-resolution, 3-D swept-source optical coherence tomography (SSOCT) to detect early airway injury changes following smoke inhalation exposure in a rabbit model. The SSOCT system obtains 3-D helical scanning using a microelectromechanical system motor-based endoscope. Real-time 2-D data processing and image display at the speed of 20 frames/s are achieved by adopting the technique of parallel computing. Longitudinal images are reconstructed via an image processing algorithm to remove motion artifacts caused by ventilation and pulse. Quantitative analyses of tracheal airway thickness as well as thickness distribution along tracheal circumference are also performed based on the comprehensive 3-D volumetric data.
Optical coherence tomography (OCT) is an evolving noninvasive imaging modality that has been used to image the human larynx during surgical endoscopy. The design of a long gradient index (GRIN) lens-based probe capable of capturing images of the human larynx by use of swept-source OCT during a typical office-based laryngoscopy examination is presented. In vivo OCT imaging of the human larynx is demonstrated with a rate of 40 frames per second. Dynamic vibration of the vocal folds is recorded to provide not only high-resolution cross-sectional tissue structures but also vibration parameters, such as the vibration frequency and magnitude of the vocal cords, which provides important information for clinical diagnosis and treatment, as well as fundamental research of the voice itself. Office-based OCT is a promising imaging modality to study the larynx for physicians in otolaryngology.
We report a multi-wavelength digital holographic tomography system based on a
fiber-based spectral interferometer. Instead of using tunable lasers, a relatively inexpensive
broadband source was used as the light source. Hundreds of 2-D "synthesized holograms" (or object
wavefields) were obtained by transversal scanning of a probe beam within a few seconds.
Holographic images of an object volume were numerically reconstructed from each synthesized
hologram and tomographic images were obtained by superposition of all the image volumes.
We present a kind of rotational two photon mciroendoscopy for 1μm fiber
femtosecond laser. The fiber laser provide ultrashort femto-second pulses with center
wavelength at 1.034μm and repetition rate of 50MH. The rotational probe is based on
double cladding photonic crystal fiber (CD PCF) fiber, Grin lens, microprism and
rotational MEMS motor. The MEMS motor has diameter of 2.2mm and can provide
360 degree full view rotation. We experimentally show that the DC PCF fiber works
for 1μm fiber laser two photon system. Second harmonic generation (SHG) singnal
line profile of rat tail tendon and fish scale was taken with the endoscopy system.
Optical coherence tomography (OCT) is an evolving noninvasive imaging modality and has been
used to image the human larynx during surgical endoscopy. The design of a long GRIN lens based
probe capable of capturing images of the human larynx by use of swept-source OCT during a typical
office-based laryngoscopy examination is presented. In vivo OCT imaging of the human larynx is
demonstrated with 40 fame/second. Dynamic vibration of the vocal folds is recorded to provide not
only high-resolution cross-sectional tissue structures but also vibration parameters, such as the
vibration frequency and magnitude of the vocal cord, which provide important information for
clinical diagnosis and treatment, as well as in fundamental research of the voice. Office-based OCT
is a promising imaging modality to study the larynx.
Doppler Optical Coherence Tomography (DOCT) imaging of in-vivo retinal blood flow was widely studied as efforts of research community to push this technology into clinic. Spectral Doppler imaging of DOCT has been demonstrated as a quantification method of in-vivo pulsatile retinal blood flow in human eye. This technology has the all the advantages inherited from OCT comparing to Doppler ultrasound. Comparing to normal spatial-distributed color Doppler imaging of DOCT, spectral Doppler imaging can reveal more haemodynamics details on the time dimension. Although resistance index (RI) of a micro-vascular can be measured in vivo from human retina, the clinical significance of RI measurements still needs to be investigated. In vitro experiment conduced with ultrasound has demonstrated the higher vascular resistance value is associated with the higher RI measured assuming the constant compliance of vascular tube. In this study, the rodent window-chamber model (RWCM) was used as a platform to investigate the RI change as the micro-vasculature response to laser irradiation. The higher RI was measured after the occlusion of two veins (should it be arterials) that was verified with laser speckle imaging in our preliminary experiment results.
Optical coherence tomography (OCT) is an evolving noninvasive imaging modality and
has been used to image the human larynx during surgical endoscopy. The design of a long GRIN
lens based probe capable of capturing images of the human larynx by use of a swept-source OCT
during a typical office-based laryngoscopy examination is presented. An optical-ballast-based 4F
optical relay system is proposed to realize variable working distance with a constant optical delay. In
vivo OCT imaging of the human larynx is demonstrated with 40 fame/second. Office-based OCT is a
promising imaging modality to study the larynx.
Digital holographic microscopy allows determination of dynamic changes in the optical thickness
profile of a transparent object with sub-wavelength accuracy. Here, we report a quantitative phase
laser microsurgery system, which takes advantage of the precise optical manipulation by the laser
microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and
temporal resolution. This system would enable absolute quantitation of localized alteration/damage
to transparent phase objects, such as the cell membrane or intra-cellular structures, being exposed to
the laser microbeam, which was not possible using conventional phase-contrast microscopy.
The laser microbeam uses lasers to alter and/or to ablate intracellular organelles and cellular and
tissue samples, and, today, has become an important tool for cell biologists to study the molecular
mechanism of complex biological systems by removing individual cells or sub-cellular organelles.
However, absolute quantitation of the localized alteration/damage to transparent phase objects, such
as the cell membrane or chromosomes, was not possible using conventional phase-contrast or
differential interference contrast microscopy. We report the development of phase-contrast digital
holographic microscopy for quantitative evaluation of cell dynamic changes in real time during laser
microsurgery. Quantitative phase images are recorded during the process of laser microsurgery and
thus, the dynamic change in phase can be continuously evaluated. Out-of-focus organelles are
re-focused by numerical reconstruction algorithms.
Optical coherence tomography (OCT) is an evolving noninvasive imaging modality that has been used to image the human larynx during surgical endoscopy. The design of a long gradient-index lens-based probe capable of capturing images of the human larynx by use of spectral domain OCT during a typical office-based laryngoscopy examination is presented. An optical-ballast-based 4f optical relay system is proposed to realize variable working distance with a constant optical delay. In-vivo OCT imaging of the human larynx is demonstrated. Office-based OCT is a promising imaging modality for early laryngeal cancer diagnosis.
The ability to inject exogenous material as well as to alter subcellular structures in a minimally invasive manner using a laser microbeam has been useful for cell biologists to study the structure-function relationship in complex biological systems. We describe a quantitative phase laser microsurgery system, which takes advantage of the combination of laser microirradiation and short-coherence interference microscopy. Using this method, quantitative phase images and the dynamic changes of phase during the process of laser microsurgery of red blood cells (RBCs) can be evaluated in real time. This system would enable absolute quantitation of localized alteration/damage to transparent phase objects, such as the cell membrane or intracellular structures, being exposed to the laser microbeam. Such quantitation was not possible using conventional phase-contrast microscopy.
A functional Fourier domain optical coherence tomography instrument offering spectral Doppler imaging of in vivo pulsatile human retinal blood flow was constructed. An improved phase-resolved algorithm was developed to correct bulk motion artifacts. Spectral Doppler imaging provides complementary temporal flow information to the spatially distributed flow information of the color Doppler image by providing direct visualization of the Doppler spectrum of the flow whose pattern can be further quantified with various velocity envelope curves and their corresponding flow indices. The coefficient of repeatability on resistance index measurement was assessed by analyzing 14 measurements on two vessels within two normal subjects.
KEYWORDS: Digital signal processing, Optical coherence tomography, Endoscopes, Signal processing, Microelectromechanical systems, 3D image processing, Image processing, Coherence imaging, 3D acquisition, Signal detection
A fast-scan-rate swept laser for optical coherence tomography (OCT) is suitable to record and analyze a 3-D image volume. However, the whole OCT system speed is limited by data streaming, processing, and storage. In this case, postprocessing is a common technique. Endoscopic clinical applications prefer onsite diagnosis, which requires a real-time technique. Parallel digital signal processors were applied to stream and process data directly from a data digitizer. A real-time system with 20-kHz axial line speed, which was limited only by our swept laser scan rate, was implemented. To couple with the system speed, an endoscope based on an improved 3-D microelectromechanical motor (diameter 1.5 mm, length 9.4 mm) was developed. In vivo 3-D imaging of the human airway was demonstrated.
Spectral domain optical coherence tomography (SDOCT) images have been used to investigate the mechanism of optical clearing in flowing blood using dextrans. The depth reflectivity profiles from SDOCT indicate that dextrans become increasingly more effective in reducing scattering in flowing blood, except for 5 mgdl−1 of Dx500, with increasing molecular weights (MW 70,000 and 500,000) and concentrations (0.6, 2, and 5 mgdl−1). Among the tested dextrans, Dx500 at 2 mgdl−1 had the most significant effect on light scattering reduction with the strongest capability to induce erythrocyte aggregation. Dx500 at 5 mgdl−1 contributes more refractive index matching but induces a decrease in aggregation that leads to the same level as 0.6 mgdl−1 Dx500. Previous studies identified various mechanisms of light scattering reduction in stationary blood induced by optical clearing agents. Our results suggest that erythrocyte aggregation is a more important mechanism for optical clearing in flowing blood using dextrans, providing a rational design basis for effective flowing blood optical clearing, which is essential for improving OCT imaging capability through flowing blood.
In this presentation, we demonstrate a novel optical tomography technique, thermoelastic
optical Doppler tomography (ODT). Short laser pulses are used to generate thermoelastic
waves in biological samples. Optical phase variations in response to wave propagation
are detected using ODT. It is shown that areas of different elastic property in the phantom
can be clearly resolved.
We propose a novel method for high accurate three-dimensional angle measurement based on
propagation vector analysis of digital holography. 3D rotations in space can be achieved by use of a
CCD camera and a multi-facet object, which reflects an incident wave into different directions. The
propagation vectors of the reflected waves from the object can be extracted by analyzing the object
spectrum of the recorded hologram. Any small rotation of the object will induce the change of the
propagation vectors in space, which can then be used for 3D angle measurement. Experimental
results are presented to verify the idea.
We present experimental demonstration of a novel full-color three-dimensional microscopy method. A white-light high-brightness LED illuminates a Michelson interferometer. A color CCD camera records the color image of an object in superposition with the reference beam. The short-coherence interferometric process is applied to each of the three color channels to extract tomographic contour images of the object. The set of three images are recombined as RGB channels to construct a color tomographic image of a section of the object. The reference mirror is scanned over a range of axial distances to construct a three-dimensional tomographic image of the object with full natural color representation. The technique is applied to imaging of artificial color objects as well as colorful biological objects with about 10 um axial resolution, about 100 ~ 200 um penetration depth, and 50 ~ 60 db dynamic range. In contrast to most existing three-dimensional microscopy methods, the present technique allows monitoring of tissue structures close to its natural color, which may be useful in various physiological and pathological applications.
KEYWORDS: Holograms, Reconstruction algorithms, 3D image reconstruction, Digital holography, Near field diffraction, Diffraction, Computer simulations, Optical simulations, Charge-coupled devices, 3D vision
This paper describes a method of numerical reconstruction for digital holography with changed viewing angles within the Fresnel domain. The proposed algorithm could not only reconstruct the real image of the original object with different focal lengths but also with changed viewing angles, which could be selected as any angle in space. Some representative simulations are presented to validate the proposed algorithm.
This paper describes a method of numerical reconstruction for digital holography with changed viewing angles within the Fresnel domain. The proposed algorithm could not only render the real image of the original object with different focal lengths but also with changed viewing angles selected in 3D space. The numerical reconstruction can be implemented by using the Fast Fourier Transform. Some representative simulations are presented to validate the proposed algorithm.
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