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PURPOSE: Subcutaneously or intramuscularly administered biodegradable microsphere formulations have been successfully exploited in the management of chronic conditions for over two decades, yet mechanistic understanding of the impact of formulation attributes on in vivo absorption rate from such systems is still in its infancy. METHODS: Suspension formulation physicochemical attributes may impact particulate deposition in subcutaneous (s.c.) tissue. Hence, the utility of synchrotron X-ray micro-computed tomography (µCT) for assessment of spatial distribution of suspension formulation components (PLG microspheres and vehicle) was evaluated in a porcine s.c. tissue model. Optical imaging of dyed vehicle and subsequent microscopic assessment of microsphere deposition was performed in parallel to compare the two approaches. RESULTS: Our findings demonstrate that synchrotron µCT can be applied to the assessment of microsphere and vehicle distribution in s.c. tissue, and that microspheres can also be visualised in the absence of contrast agent using this approach. The technique was deemed superior to optical imaging of macrotomy for the characterisation of microsphere deposition owing to its non-invasive nature and relatively rapid data acquisition time. CONCLUSIONS: The method outlined in this study provides a proof of concept feasibility for µCT application to determining the vehicle and suspended PLG microspheres fate following s.c. injection. A potential application for our findings is understanding the impact of injection, device and formulation variables on initial and temporal depot geometry in pre-clinical or ex-vivo models that can inform product design. Graphical abstract.
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Materiais Biocompatíveis/química , Meios de Contraste/química , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Suspensões/química , Tomografia Computadorizada por Raios X/métodos , Animais , Composição de Medicamentos , Imageamento Tridimensional , Injeções Subcutâneas , Intensificação de Imagem Radiográfica , Suínos , Síncrotrons , Alicerces Teciduais/químicaRESUMO
Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.
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Processamento de Imagem Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , HumanosRESUMO
Small angle x-ray scattering has been proven to be a valuable method for accessing structural information below the spatial resolution limit implied by direct imaging. Here, we theoretically derive the relation that links the subpixel differential phase signal provided by the sample to the moments of scattering distributions accessible by refraction sensitive x-ray imaging techniques. As an important special case we explain the scatter or dark-field contrast in terms of the sample's phase signal. Further, we establish that, for binary phase objects, the nth moment scales with the difference of the refractive index decrement to the power of n. Finally, we experimentally demonstrate the utility of the moments by quantitatively determining the particle sizes of a range of powders with a laboratory-based setup.
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X-ray phase contrast enables weakly-attenuating structures to be imaged, with bright synchrotron sources adding the ability to capture time sequences and analyse sample dynamics. Here, we describe the translation of dynamical differential phase contrast imaging from the synchrotron to a compact x-ray source, in order to achieve this kind of time sequence imaging in the laboratory. We formulate broadly-applicable set-up guidelines for the single-grid, single-exposure imaging technique using a divergent source, exploring the experimental factors that restrict set-up size, imaging sensitivity and sample size. Experimental images are presented using the single-grid phase contrast technique with a steel attenuation grid and a liquid-metal-jet x-ray source, enabling exposure times as short as 0.5 s for dynamic imaging. Differential phase contrast images were retrieved from phantoms, incorporating noise filtering to improve the low-count images encountered when imaging dynamics using short exposures.
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Laboratórios , Imagem Molecular/métodos , Imagem Molecular/instrumentação , Imagens de Fantasmas , Razão Sinal-Ruído , Síncrotrons , Raios XRESUMO
Data-constrained modeling is a method that enables three-dimensional distribution of mineral phases and porosity in a sample to be modeled based on micro-computed tomography scans acquired at different X-ray energies. Here we describe an alternative method for measuring porosity, synchrotron K-edge subtraction using xenon gas as a contrast agent. Results from both methods applied to the same Darai limestone sample are compared. Reasonable agreement between the two methods and with other porosity measurements is obtained. The possibility of a combination of data-constrained modeling and K-edge subtraction methods for more accurate sample characterization is discussed.
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Floods can completely submerge terrestrial plants but some wetland species can sustain O2 and CO2 exchange with the environment via gas films forming on superhydrophobic leaf surfaces. We used high resolution synchrotron X-ray phase contrast micro-tomography in a novel approach to visualise gas films on submerged leaves of common cordgrass (Spartina anglica). 3D tomograms enabled a hitherto unmatched level of detail regarding the micro-topography of leaf gas films. Gas films formed only on the superhydrophobic adaxial leaf side (water droplet contact angle, Φ=162°) but not on the abaxial side (Φ=135°). The adaxial side of the leaves of common cordgrass is plicate with a longitudinal system of parallel grooves and ridges and the vast majority of the gas film volume was found in large â¼180µm deep elongated triangular volumes in the grooves and these volumes were connected to each neighbouring groove via a fine network of gas tubules (â¼1.7µm diameter) across the ridges. In addition to the gas film retained on the leaf exterior, the X-ray phase contrast micro-tomography also successfully distinguished gas spaces internally in the leaf tissues, and the tissue porosity (gas volume per unit tissue volume) ranged from 6.3% to 20.3% in tip and base leaf segments, respectively. We conclude that X-ray phase contrast micro-tomography is a powerful tool to obtain quantitative data of exterior gas features on biological samples because of the significant difference in electron density between air, biological tissues and water.
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Dióxido de Carbono/metabolismo , Oxigênio/metabolismo , Fotossíntese , Folhas de Planta/ultraestrutura , Dióxido de Carbono/química , Meio Ambiente , Interações Hidrofóbicas e Hidrofílicas , Oxigênio/química , Folhas de Planta/química , Poaceae/química , Poaceae/ultraestrutura , Síncrotrons , Tomografia , Água/química , Raios XRESUMO
Phase contrast x-ray imaging (PCXI) is a promising imaging modality, capable of sensitively differentiating soft tissue structures at high spatial resolution. However, high sensitivity often comes at the cost of a long exposure time or multiple exposures per image, limiting the imaging speed and possibly increasing the radiation dose. Here, we demonstrate a PCXI method that uses a single short exposure to sensitively capture sample phase information, permitting high speed x-ray movies and live animal imaging. The method illuminates a checkerboard phase grid to produce a fine grid-like intensity reference pattern at the detector, then spatially maps sample-induced distortions of this pattern to recover differential phase images of the sample. The use of a phase grid is an improvement on our previous absorption grid work in two ways. There is minimal loss in x-ray flux, permitting faster imaging, and, a very fine pattern is produced for homogenous high spatial resolution. We describe how this pattern permits retrieval of five images from a single exposure; the sample phase gradient images in the horizontal and vertical directions, a projected phase depth image, an edge-enhanced image, and a type of scattering image. Finally, we describe how the reconstruction technique can achieve subpixel distortion retrieval and study the behavior of the technique in regard to analysis technique, Talbot distance, and exposure time.
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Iluminação/instrumentação , Microscopia de Contraste de Fase/instrumentação , Refratometria/instrumentação , Transdutores , Difração de Raios X/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Newly developed high-speed, synchrotron-based X-ray computed microtomography enabled us to directly image pore-scale displacement events in porous rock in real time. Common approaches to modeling macroscopic fluid behavior are phenomenological, have many shortcomings, and lack consistent links to elementary pore-scale displacement processes, such as Haines jumps and snap-off. Unlike the common singular pore jump paradigm based on observations of restricted artificial capillaries, we found that Haines jumps typically cascade through 10-20 geometrically defined pores per event, accounting for 64% of the energy dissipation. Real-time imaging provided a more detailed fundamental understanding of the elementary processes in porous media, such as hysteresis, snap-off, and nonwetting phase entrapment, and it opens the way for a rigorous process for upscaling based on thermodynamic models.
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Rats display little to no haversian remodeling of cortical bone. This fact, combined with the endochondral formation of cortical bone, means that rat femoral cortical bone contains highly mineralized cartilage islands in a central band of mid-femoral cross sections. We demonstrate that these islands have a significantly higher degree of mineralization than the surrounding bone, using quantitative backscattered electron imaging. The cartilaginous nature of the islands was verified by immunostaining for collagen type II. Toluidine blue staining of longitudinal sections and three-dimensional synchrotron radiation X-ray tomographic microscopy confirmed that the islands are elongated along the femoral long axis. Nanoindentation revealed significantly higher values of both reduced modulus and hardness in the islands compared to the surrounding bone, reflecting a higher degree of mineralization. The calcified cartilage islands were distributed in a central zone of the bone, from the growth plates through the mid-femoral bone. The presence of these cartilage islands and their possible effect on mechanical properties could be an additional reason why haversian remodeling is observed in higher-order species.
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Calcificação Fisiológica/fisiologia , Cálcio/metabolismo , Cartilagem/metabolismo , Fêmur/metabolismo , Agrecanas/metabolismo , Animais , Colágeno Tipo II/metabolismo , Colágeno Tipo XI/metabolismo , Feminino , Fêmur/citologia , Histocitoquímica , Ratos , Ratos Wistar , Microtomografia por Raio-XRESUMO
The purpose of this study was to investigate a selection of psychological variables (help-seeking behaviors, mental imagery, self-esteem) in relation to injury among UK dancers. We recruited 216 participants from eight dance styles and six levels of involvement. It was found that 83.5% of the participants had experienced at least one injury in the past year. The most common response to injury was to inform someone, and most continued to dance when injured, albeit carefully. Physical therapy was the most common treatment sought when an injury occurred (38.1%), and dancers seemed to follow recommendations offered. Injured and non-injured dancers did not differ in their imagery frequencies (facilitative, debilitative, or injury-related) and scored similarly (and relatively high) in self-esteem. Neither facilitative nor debilitative imagery was correlated with self-esteem, but dancers who engaged in more facilitative imagery in general also reported doing so when injured. Altogether, it appears that injury is not related to dancers' self-esteem or imagery, at least not when injuries are mild or moderate. Even so, such conclusions should be made with caution, given that most dancers do sustain at least one injury each year.
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Dança/lesões , Dança/psicologia , Autoimagem , Adolescente , Adulto , Feminino , Humanos , Imaginação , Masculino , Pessoa de Meia-Idade , Aceitação pelo Paciente de Cuidados de Saúde/psicologia , Modalidades de Fisioterapia , Autoeficácia , Adulto JovemRESUMO
We present a time-resolved tomographic reconstruction of the velocity field associated with pulsatile blood flow through a rotationally-symmetric stenotic vessel model. The in-vitro sample was imaged using propagation-based phase contrast with monochromated X-rays from a synchrotron undulator source, and a fast shutter-synchronized detector with high-resolution used to acquire frames of the resulting dynamic speckle pattern. Having used phase retrieval to decode the phase contrast from the speckle patterns, the resulting projected-density maps were analysed using the statistical correlation methods of particle image velocimetry (PIV). This yields the probability density functions of blood-cell displacement within the vessel. The axial velocity-field component of the rotationally-symmetric flow was reconstructed using an inverse-Abel transform. A modified inverse-Abel transform was used to reconstruct the radial component. This vector tomographic phase-retrieval velocimetry was performed over the full pumping cycle, to completely characterize the velocity field of the pulsatile blood flow in both space and time.
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Fluxo Pulsátil/fisiologia , Reologia/métodos , Tomografia por Raios X/métodos , Análise de FourierRESUMO
While there is currently a validated dance-specific exercise method of measuring aerobic fitness, no such test has been developed to measure high intensity capabilities in dance. The purpose of this study was to initiate an intermittent high intensity dance-specific fitness test. The test was designed to be able to observe changes in heart rate (HR), thereby allowing for a measurement of physical fitness at high intensities. Sixteen professional dancers (4 males and 12 females) volunteered to take part in this study. The fitness test protocol consists of movements that are representative of contemporary dance, and contains exercise and rest periods that mimic the intermittent nature of dance. The participants performed four trials. The physiological variables measured were HR (b.min(-1)) for each one minute bout of the four minute test for all trials, oxygen uptake (VO(2)) throughout the test, and end blood lactate (BLa mmol.L) for each trial. In addition, five of the participants undertook a maximal oxygen uptake treadmill test, and the scores obtained were compared with those from the dance test. Results show HR consistency across each one minute bout of the test and across each of the four trials of testing for all participants, indicating that the test is reliable. There was good reliability between bouts of each trial (typical error as % of CV = 1.5), intraclass "r" = 0.8, and good reliability between the four trials (typical error as % of CV = 2.1), intraclass "r" = 0.82. There were no significant differences between the maximal VO(2) and BLa scores established in the treadmill and dance tests, demonstrating validity. Thus, the results of this study indicate that the high intensity dance-specific test is a reliable and valid means of assessing and monitoring the cardiovascular fitness of dancers. The test allows dancers to be assessed within an environment that they are accustomed to (the studio), using a mode of exercise that is relevant (dance), and it is of adequate intensity to be representative of performance.
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Dança/fisiologia , Teste de Esforço , Exercício Físico/fisiologia , Consumo de Oxigênio/fisiologia , Esforço Físico/fisiologia , Aptidão Física/fisiologia , Adulto , Análise de Variância , Teste de Esforço/métodos , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
Aeration of the lung and the transition to air-breathing at birth is fundamental to mammalian life and initiates major changes in cardiopulmonary physiology. However, the dynamics of this process and the factors involved are largely unknown, because it has not been possible to observe or measure lung aeration on a breath-by-breath basis. We have used the high contrast and spatial resolution of phase contrast X-ray imaging to study lung aeration at birth in spontaneously breathing neonatal rabbits. As the liquid-filled fetal lungs provide little absorption or phase contrast, they are not visible and only become visible as they aerate, allowing a detailed examination of this process. Pups were imaged live from birth to determine the timing and spatial pattern of lung aeration, and relative levels of lung aeration were measured from the images using a power spectral analysis. We report the first detailed observations and measurements of lung aeration, demonstrating its dependence on inspiratory activity and body position; dependent regions aerated at much slower rates. The air/liquid interface moved toward the distal airways only during inspiration, with little proximal movement during expiration, indicating that trans-pulmonary pressures play an important role in airway liquid clearance at birth. Using these imaging techniques, the dynamics of lung aeration and the critical role it plays in regulating the physiological changes at birth can be fully explored.