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1.
ACS Sens ; 4(2): 456-463, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30644723

RESUMO

Bioreactors have been used both to develop new, and to improve bioprocess yields for, biopharmaceutical products. However, efforts to miniaturize bioreactors, in order to save costs and accelerate process development times, have been limited by the lack of on-site monitoring capabilities available at such scales. In this study, small volume (3 nL) nonconsumptive holographic sensors were integrated into a glass-PDMS microfluidic chip to monitor via a blue-shift in the resultant holographic replay wavelength, the change in pH during microbial growth of Lactobacillus casei ( L. casei) Shirota. Within the optimal growth pH range of L. casei, the accuracy of the miniaturized pH sensors was comparable to that of a conventional pH meter. Conceivably, this approach could be extrapolated to an array of miniaturized holographic sensors sensitive to different analytes, and thereby paving the way for reliable, real-time, noninvasive monitoring of microorganisms in a nanobioreactor.


Assuntos
Holografia/instrumentação , Dispositivos Lab-On-A-Chip , Lactobacillus casei/crescimento & desenvolvimento , Acetona/análogos & derivados , Acetona/química , Dimetilpolisiloxanos/química , Concentração de Íons de Hidrogênio , Metacrilatos/química , Nylons/química
2.
Analyst ; 144(5): 1751-1760, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30666996

RESUMO

The accurate and fast size classification of microparticles is important in environmental monitoring and biomedical applications. Conventional methods for sensing and classifying microparticles require bulky optical setups and generally show medium performance. Accordingly, the development of a portable and smart platform for accurate particle size classification is essential. In this study, we propose a new sensing platform for automatic identification of microparticle types through the synergistic integration of smartphone-based digital in-line holographic microscopy (DIHM) and machine-learning algorithms. The smartphone-based DIHM system consists of a coherent laser beam, a pinhole, a sample holder, a three-dimensional printed attachment, and a modified built-in smartphone camera module. The portable device has a physical dimension of 4 × 8 × 10 cm3 and 220 g in weight. Holograms of various polystyrene microparticles with different sizes (d = 2-50 µm) were recorded with a wide field-of-view and high spatial resolution. To establish a proper classification model, tens of features including geometrical parameters and light-intensity distributions were extracted from holograms of individual particles, and five machine-learning algorithms were used. After examining the performance of several classifiers, the resulting support vector machine model trained by using three geometrical parameters and three extracted parameters from light-intensity distributions shows the highest accuracy in the particle classification of the training and test sets (>98%). Therefore, the developed handheld smartphone-based platform can be potentially utilized to cope with various imaging needs in mobile healthcare and environmental monitoring.


Assuntos
Holografia/instrumentação , Microscopia/instrumentação , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/classificação , Smartphone , Algoritmos , Holografia/métodos , Aprendizado de Máquina , Microscopia/métodos
3.
J Biomed Opt ; 23(11): 1-8, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30444085

RESUMO

Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.


Assuntos
Holografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Células COS , Desenho de Equipamento , Holografia/instrumentação , Lasers , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
4.
Opt Express ; 26(19): 25211-25225, 2018 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-30469626

RESUMO

We present a new approach for combining holographic optical tweezers with confocal Raman spectroscopy. Multiple laser foci, generated using a liquid-crystal spatial light modulator, are individually used for both optical trapping and excitation of spontaneous Raman spectroscopy from trapped objects. Raman scattering from each laser focus is spatially filtered using reflective apertures on a digital micro-mirror device, which can be reconfigured with flexible patterns at video rate. We discuss operation of the instrument, and performance and viability considerations for biological measurements. We then demonstrate the capability of the instrument for fast, flexible, and interactive manipulation with molecular measurement of interacting live cell systems.


Assuntos
Bactérias/citologia , Células Dendríticas/citologia , Holografia/instrumentação , Pinças Ópticas , Análise Espectral Raman/instrumentação , Linfócitos T/citologia , Desenho de Equipamento , Luz
5.
Opt Express ; 26(20): 26520-26533, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30469737

RESUMO

For the see-through and near-to-eye displays, light throughput and uniformity of luminance over the field of view are improved by employing an optical image guide with discretely depth-varying surface relief holographic gratings. In the design process, a newly developed mathematical model, in conjunction with rigorous coupled wave analysis of diffraction efficiency, eliminates massive and time consuming iteration of non-sequential ray tracing but rapidly identifies the depth-varying structure and optimum optical performance. The depth-varying grating based approach achieved a 1.37x improvement in light throughput compared to the conventional depth un-varying design, 315 cd/m2/lm, along with improved uniformity over the field of view of 35 (H) x 20 (V) degrees with an eye box size of 17 (H) x 14 (V) mm.


Assuntos
Simulação por Computador , Holografia/instrumentação , Modelos Teóricos , Algoritmos , Desenho de Equipamento , Holografia/métodos , Processamento de Imagem Assistida por Computador , Refratometria/métodos
6.
Sensors (Basel) ; 18(11)2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-30423978

RESUMO

The authors recently developed a two-dimensional (2D) holographic electromagnetic induction imaging (HEI) for biomedical imaging applications. However, this method was unable to detect small inclusions accurately. For example, only one of two inclusions can be detected in the reconstructed image if the two inclusions were located at the same XY plane but in different Z-directions. This paper provides a theoretical framework of three-dimensional (3D) HEI to accurately and effectively detect inclusions embedded in a biological object. A numerical system, including a realistic head phantom, a 16-element excitation sensor array, a 16-element receiving sensor array, and image processing model has been developed to evaluate the effectiveness of the proposed method for detecting small stroke. The achieved 3D HEI images have been compared with 2D HEI images. Simulation results show that the 3D HEI method can accurately and effectively identify small inclusions even when two inclusions are located at the same XY plane but in different Z-directions. This preliminary study shows that the proposed method has the potential to develop a useful imaging tool for the diagnosis of neurological diseases and injuries in the future.


Assuntos
Encéfalo/diagnóstico por imagem , Diagnóstico por Imagem/métodos , Holografia/instrumentação , Acidente Vascular Cerebral/diagnóstico por imagem , Algoritmos , Encéfalo/patologia , Radiação Eletromagnética , Humanos , Processamento de Imagem Assistida por Computador , Imagem Tridimensional , Imagens de Fantasmas , Acidente Vascular Cerebral/patologia
7.
Otol Neurotol ; 39(10): e1137-e1142, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30239435

RESUMO

OBJECTIVES: A mixed reality (MR) headset that enables three-dimensional (3D) visualization of interactive holograms anchored to specific points in physical space was developed for use with lateral skull base anatomy. The objectives of this study are to: 1) develop an augmented reality platform using the headset for visualization of temporal bone structures, and 2) measure the accuracy of the platform as an image guidance system. METHODS: A combination of semiautomatic and manual segmentation was used to generate 3D reconstructions of soft tissue and bony anatomy of cadaver heads and temporal bones from 2D computed tomography images. A Mixed-Reality platform was developed using C# programming to generate interactive 3D holograms that could be displayed in the HoloLens headset. Accuracy of visual surface registration was determined by target registration error between seven predefined points on a 3D holographic skull and 3D printed model. RESULTS: Interactive 3D holograms of soft tissue, bony anatomy, and internal ear structures of cadaveric models were generated and visualized in the MR headset. Software user interface was developed to allow for user control of the virtual images through gaze, voice, and gesture commands. Visual surface point matching registration was used to align and anchor holograms to physical objects. The average target registration error of our system was 5.76 mm ±â€Š0.54. CONCLUSION: In this article, we demonstrate that an MR headset can be applied to display interactive 3D anatomic structures of the temporal bone that can be overlaid on physical models. This technology has the potential to be used as an image guidance tool during anatomic dissection and lateral skull base surgery.


Assuntos
Holografia/métodos , Procedimentos Neurocirúrgicos/métodos , Base do Crânio/anatomia & histologia , Software , Cirurgia Assistida por Computador/métodos , Cadáver , Holografia/instrumentação , Humanos , Procedimentos Neurocirúrgicos/instrumentação , Base do Crânio/cirurgia , Cirurgia Assistida por Computador/instrumentação , Tomografia Computadorizada por Raios X
8.
Cytometry A ; 93(10): 987-996, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30211977

RESUMO

Last decade's advancements in optofluidics allowed obtaining an ever increasing integration of different functionalities in lab on chip devices to culture, analyze, and manipulate single cells and entire biological specimens. Despite the importance of optical imaging for biological sample monitoring in microfluidics, imaging is traditionally achieved by placing microfluidics channels in standard bench-top optical microscopes. Recently, the development of either integrated optical elements or lensless imaging methods allowed optical imaging techniques to be implemented in lab on chip systems, thus increasing their automation, compactness, and portability. In this review, we discuss known solutions to implement microscopes on chip that exploit different optical methods such as bright-field, phase contrast, holographic, and fluorescence microscopy.


Assuntos
Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Microscopia/instrumentação , Microscopia/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Automação/instrumentação , Automação/métodos , Holografia/instrumentação , Holografia/métodos , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação
9.
Opt Lett ; 43(17): 4096-4099, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30160725

RESUMO

The implementation of a numerical dark field illumination to obtain reconstructions with enhanced contrast in digital lensless holographic (DLHM) microscopy is presented. In this proposal, an opaque circular stop is numerically inserted in the light path between the sample and hologram plane. The contrast of the reconstructed holograms is increased at least 46% in comparison with that obtained by the regular reconstruction methods. This proposal has been validated using experimental holograms of a paramecium in water and a section of the head of a Drosophila melanogaster fly recorded in DLHM.


Assuntos
Holografia/instrumentação , Iluminação/métodos , Microscopia/instrumentação , Razão Sinal-Ruído , Animais , Drosophila melanogaster
10.
Sci Rep ; 8(1): 12020, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104699

RESUMO

Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.


Assuntos
Movimento Celular , Fibrossarcoma/patologia , Microscopia Intravital/métodos , Invasividade Neoplásica/diagnóstico por imagem , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colágeno/metabolismo , Fibrossarcoma/diagnóstico por imagem , Holografia/instrumentação , Holografia/métodos , Humanos , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Microscopia Intravital/instrumentação , Invasividade Neoplásica/patologia , Pseudópodes/metabolismo
11.
J Biophotonics ; 11(11): e201800010, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29920960

RESUMO

Optical sectioning endoscopy such as confocal endoscopy offers capabilities to obtain three-dimensional (3D) information from various biological samples by discriminating between the desired in-focus signals and out-of-focus background. However, in general confocal images are formed through point-by-point scanning and the scanning time is proportional to the 3D space-bandwidth product. Recently, structured illumination endoscopy has been utilized for optically sectioned wide-field imaging, but it still needs axial scanning to acquire images from different depths of focal plane. Here, we report wide-field, multiplane, optical sectioning endoscopic imaging, incorporating 3D active speckle-based illumination and multiplexed volume holographic gratings, to simultaneously obtain images of fluorescently labeled tissue structures from different depths, without the need of scanning. We present the design, and implementation, as well as experimental data, demonstrating this endoscopic system's ability to obtain optically sectioned multiplane fluorescent images of tissue samples, with cellular level resolution in wide-field fashion, and no need for mechanical or optical axial scanning.(A) Schematic drawing of the SIHN endoscopy to simultaneously acquire multiplane images from different depths. (B) Uniform, and (C) SIHN illuminated images of standard fluorescence beads (25 µm in diameter) for the two axial planes. (D) Intensity profile on fluorescently labeled signal (ie, in-focus) and background (ie, out-of-focus) of microspheres.


Assuntos
Endoscopia/métodos , Fluorescência , Holografia/métodos , Desenho de Equipamento , Holografia/instrumentação
12.
Opt Lett ; 43(9): 1943-1946, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29714767

RESUMO

We present an external interferometric setup that is able to simultaneously acquire three wavelengths of the same sample instance without scanning or multiple exposures. This setup projects onto the monochrome digital camera three off-axis holograms with rotated fringe orientations, each from a different wavelength channel, without overlap in the spatial-frequency domain, and thus allows the full reconstruction of the three complex wavefronts from the three wavelength channels. We use this new setup for three-wavelength phase unwrapping, allowing phase imaging of thicker objects than possible with a single wavelength, but without the increased level of noise. We demonstrate the proposed technique for micro-channel profiling and label-free cell imaging.


Assuntos
Holografia/instrumentação , Imagem Tridimensional/instrumentação , Melanoma/diagnóstico por imagem , Desenho de Equipamento , Holografia/métodos , Humanos , Aumento da Imagem/métodos , Imagem Tridimensional/métodos , Melanoma/patologia , Células Tumorais Cultivadas
13.
ACS Nano ; 12(3): 2554-2559, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29522316

RESUMO

We present a cost-effective and portable platform based on contact lenses for noninvasively detecting Staphylococcus aureus, which is part of the human ocular microbiome and resides on the cornea and conjunctiva. Using S. aureus-specific antibodies and a surface chemistry protocol that is compatible with human tears, contact lenses are designed to specifically capture S. aureus. After the bacteria capture on the lens and right before its imaging, the captured bacteria are tagged with surface-functionalized polystyrene microparticles. These microbeads provide sufficient signal-to-noise ratio for the quantification of the captured bacteria on the contact lens, without any fluorescent labels, by 3D imaging of the curved surface of each lens using only one hologram taken with a lens-free on-chip microscope. After the 3D surface of the contact lens is computationally reconstructed using rotational field transformations and holographic digital focusing, a machine learning algorithm is employed to automatically count the number of beads on the lens surface, revealing the count of the captured bacteria. To demonstrate its proof-of-concept, we created a field-portable and cost-effective holographic microscope, which weighs 77 g, controlled by a laptop. Using daily contact lenses that are spiked with bacteria, we demonstrated that this computational sensing platform provides a detection limit of ∼16 bacteria/µL. This contact-lens-based wearable sensor can be broadly applicable to detect various bacteria, viruses, and analytes in tears using a cost-effective and portable computational imager that might be used even at home by consumers.


Assuntos
Lentes de Contato/microbiologia , Holografia/instrumentação , Aprendizado de Máquina , Microscopia/instrumentação , Staphylococcus aureus/isolamento & purificação , Desenho de Equipamento , Holografia/métodos , Humanos , Microscopia/métodos , Infecções Estafilocócicas/microbiologia
14.
Comput Biol Med ; 96: 147-156, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29573668

RESUMO

Three-part white blood cell differentials which are key to routine blood workups are typically performed in centralized laboratories on conventional hematology analyzers operated by highly trained staff. With the trend of developing miniaturized blood analysis tool for point-of-need in order to accelerate turnaround times and move routine blood testing away from centralized facilities on the rise, our group has developed a highly miniaturized holographic imaging system for generating lens-free images of white blood cells in suspension. Analysis and classification of its output data, constitutes the final crucial step ensuring appropriate accuracy of the system. In this work, we implement reference holographic images of single white blood cells in suspension, in order to establish an accurate ground truth to increase classification accuracy. We also automate the entire workflow for analyzing the output and demonstrate clear improvement in the accuracy of the 3-part classification. High-dimensional optical and morphological features are extracted from reconstructed digital holograms of single cells using the ground-truth images and advanced machine learning algorithms are investigated and implemented to obtain 99% classification accuracy. Representative features of the three white blood cell subtypes are selected and give comparable results, with a focus on rapid cell recognition and decreased computational cost.


Assuntos
Citometria de Fluxo/métodos , Holografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Leucócitos/citologia , Análise de Célula Única/métodos , Algoritmos , Desenho de Equipamento , Citometria de Fluxo/instrumentação , Holografia/instrumentação , Humanos , Aprendizado de Máquina , Miniaturização , Análise de Célula Única/instrumentação
15.
Appl Opt ; 57(1): A242-A249, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29328152

RESUMO

A single-shot water-immersion digital holographic microscope combined with broadband (white light) illumination mode is presented. This double imaging platform allows conventional incoherent visualization with phase holographic imaging of inspected samples. The holographic architecture is implemented at the image space (that is, after passing the microscope lens), thus reducing the sensitivity of the system to vibrations and/or thermal changes in comparison to regular interferometers. Because of the off-axis holographic recording principle, quantitative phase images of live biosamples can be recorded in a single camera snapshot at full-field geometry without any moving parts. And, the use of water-immersion imaging lenses maximizes the achievable resolution limit. This dual-mode microscope platform is first calibrated using microbeads, then applied to the characterization of fixed cells (neuroblastoma, breast cancer, and hippocampal neuronal cells) and, finally, validated for visualization of dynamic living cells (hippocampal neurons).


Assuntos
Holografia/métodos , Iluminação/métodos , Microscopia/métodos , Calibragem , Desenho de Equipamento , Holografia/instrumentação , Imersão , Microscopia/instrumentação
17.
Opt Lett ; 42(23): 4841-4844, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29216124

RESUMO

In this Letter, the use of two-coupled Mach-Zehnder interferometers for four π/2-phase shifting interferometry is introduced. A multi-camera arrangement using no more than beam splitters and mirrors is utilized to obtain in a single shot the needed phase-shifted interferograms in the different output channels of the setup. The simplicity of the setup makes it ideal for high-speed interferometry applications. This proposal is validated in digital holographic microscopy to visualize a biological sample of epidermal onion cells.


Assuntos
Holografia/instrumentação , Microscopia/instrumentação , Desenho de Equipamento , Interferometria/instrumentação
18.
J Biomed Opt ; 22(10): 1-4, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28971662

RESUMO

A wavelength-coded volume holographic imaging (WC-VHI) endoscope system capable of simultaneous multifocal imaging is presented. The system images light from two depths separated by 100 µm in a tissue sample by using axial chromatic dispersion of a gradient index probe in combination with two light-emitting diode sources and a multiplexed volume hologram to separate the images. This system is different from previous VHI systems in that it uses planar multiplexed gratings and does not require curved holographic gratings. This results in improved lateral imaging resolution from 228.1 to 322.5 lp/mm. This letter describes the design and fabrication of the WC-VHI endoscope and experimental images of hard and soft resolution targets and biological tissue samples to illustrate the performance properties.


Assuntos
Endoscópios , Holografia/instrumentação
19.
Plast Reconstr Surg ; 140(5): 1066-1070, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29068946

RESUMO

Virtual reality and augmented reality devices have recently been described in the surgical literature. The authors have previously explored various iterations of these devices, and although they show promise, it has become clear that virtual reality and/or augmented reality devices alone do not adequately meet the demands of surgeons. The solution may lie in a hybrid technology known as mixed reality, which merges many virtual reality and augmented realty features. Microsoft's HoloLens, the first commercially available mixed reality device, provides surgeons intraoperative hands-free access to complex data, the real environment, and bidirectional communication. This report describes the use of HoloLens in the operating room to improve decision-making and surgical workflow. The pace of mixed reality-related technological development will undoubtedly be rapid in the coming years, and plastic surgeons are ideally suited to both lead and benefit from this advance.


Assuntos
Holografia/instrumentação , Procedimentos Cirúrgicos Reconstrutivos/instrumentação , Cirurgia Assistida por Computador/instrumentação , Interface Usuário-Computador , Tomada de Decisão Clínica , Humanos , Procedimentos Cirúrgicos Reconstrutivos/métodos , Cirurgia Assistida por Computador/métodos , Fluxo de Trabalho
20.
Sci Adv ; 3(8): e1700606, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28798957

RESUMO

Establishing early warning systems for anthrax attacks is crucial in biodefense. Despite numerous studies for decades, the limited sensitivity of conventional biochemical methods essentially requires preprocessing steps and thus has limitations to be used in realistic settings of biological warfare. We present an optical method for rapid and label-free screening of Bacillus anthracis spores through the synergistic application of holographic microscopy and deep learning. A deep convolutional neural network is designed to classify holographic images of unlabeled living cells. After training, the network outperforms previous techniques in all accuracy measures, achieving single-spore sensitivity and subgenus specificity. The unique "representation learning" capability of deep learning enables direct training from raw images instead of manually extracted features. The method automatically recognizes key biological traits encoded in the images and exploits them as fingerprints. This remarkable learning ability makes the proposed method readily applicable to classifying various single cells in addition to B. anthracis, as demonstrated for the diagnosis of Listeria monocytogenes, without any modification. We believe that our strategy will make holographic microscopy more accessible to medical doctors and biomedical scientists for easy, rapid, and accurate point-of-care diagnosis of pathogens.


Assuntos
Antraz/diagnóstico , Antraz/microbiologia , Bacillus anthracis/citologia , Aprendizado Profundo , Holografia , Microscopia , Algoritmos , Análise de Dados , Holografia/instrumentação , Holografia/métodos , Humanos , Processamento de Imagem Assistida por Computador , Aprendizado de Máquina , Microscopia/instrumentação , Microscopia/métodos , Esporos Bacterianos
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