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International Journal of Molecular... Feb 2017This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography...
This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT), the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block-DB) cultured with human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures of hPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seeded onto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained by SR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successively processed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE). The chosen experimental method, previously demonstratively applied for the 3D characterization of the same constructs in not xeno-free media, quantitatively monitored also in this case the early stages of bone formation in basal and differentiating conditions. Interestingly, it quantitatively showed in the xeno-free environment a significant acceleration of the mineralization process, regardless of the culture (basal/differentiating) medium. This work showed in 3D that the DB guides the osteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observations and functional studies previously performed by some of the authors. Indeed, here we fully proved in 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis for Good Manufacturing Practice (preserving the stem cells' morphological features and their ability to differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds, as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.
Topics: Adult; Animals; Cell Culture Techniques; Cell Differentiation; Humans; Microscopy, Phase-Contrast; Osteogenesis; Periodontal Ligament; Stem Cells; Synchrotrons; Tissue Engineering; Tissue Scaffolds
PubMed: 28208578
DOI: 10.3390/ijms18020364 -
Optics Express May 2011In-line phase-contrast X-ray imaging provides images where both absorption and refraction contribute. For quantitative analysis of these images, the phase needs to be...
In-line phase-contrast X-ray imaging provides images where both absorption and refraction contribute. For quantitative analysis of these images, the phase needs to be retrieved numerically. There are many phase-retrieval methods available. Those suitable for phase-contrast tomography, i.e., non-iterative phase-retrieval methods that use only one image at each projection angle, all follow the same pattern though derived in different ways. We outline this pattern and use it to compare the methods to each other, considering only phase-retrieval performance and not the additional effects of tomographic reconstruction. We also outline derivations, approximations and assumptions, and show which methods are similar or identical and how they relate to each other. A simple scheme for choosing reconstruction method is presented, and numerical phase-retrieval performed for all methods.
Topics: Absorption; Algorithms; Equipment Design; Materials Testing; Microscopy, Phase-Contrast; Models, Statistical; Optics and Photonics; Photons; Radiographic Image Interpretation, Computer-Assisted; Tomography, X-Ray Computed; X-Rays
PubMed: 21643293
DOI: 10.1364/OE.19.010359 -
Journal of Biomedical Optics 2008We describe how phase-modulation laser interference microscopy and wavelet analysis can be applied to noninvasive nonstained visualization and study of the structural...
We describe how phase-modulation laser interference microscopy and wavelet analysis can be applied to noninvasive nonstained visualization and study of the structural and dynamical properties of living cells. We show how phase images of erythrocytes can reveal the difference between various erythrocyte forms and stages of hemolysis and how phase images of neurons reveal their complex intracellular structure. Temporal variations of the refractive index are analyzed to detect cellular rhythmic activity on different time scales as well as to uncover interactions between the cellular processes.
Topics: Cells, Cultured; Erythrocytes; Humans; Image Enhancement; Interferometry; Microscopy, Confocal; Microscopy, Phase-Contrast; Tomography, Optical Coherence
PubMed: 18601549
DOI: 10.1117/1.2937213 -
PloS One 2013The development of phase contrast methods for diagnostic x-ray imaging is inspired by the potential of seeing the internal structures of the human body without the need...
The development of phase contrast methods for diagnostic x-ray imaging is inspired by the potential of seeing the internal structures of the human body without the need to deposit any harmful radiation. An efficient class of x-ray phase contrast imaging and scatter correction methods share the idea of using structured illumination in the form of a periodic fringe pattern created with gratings or grids. They measure the scatter and distortion of the x-ray wavefront through the attenuation and deformation of the fringe pattern via a phase stepping process. Phase stepping describes image acquisition at regular phase intervals by shifting a grating in uniform steps. However, in practical conditions the actual phase intervals can vary from step to step and also spatially. Particularly with the advent of electromagnetic phase stepping without physical movement of a grating, the phase intervals are dependent upon the focal plane of interest. We describe a demodulation algorithm for phase stepping at arbitrary and position-dependent (APD) phase intervals without assuming a priori knowledge of the phase steps. The algorithm retrospectively determines the spatial distribution of the phase intervals by a Fourier transform method. With this ability, grating-based x-ray imaging becomes more adaptable and robust for broader applications.
Topics: Algorithms; Diagnostic Imaging; Electromagnetic Phenomena; Fourier Analysis; Light; Microscopy, Phase-Contrast; Retrospective Studies; Scattering, Radiation; Tomography, X-Ray Computed; X-Rays
PubMed: 24205177
DOI: 10.1371/journal.pone.0078276 -
Optics Express Sep 2010We have developed two phase-retrieval techniques for analyser-based phase contrast imaging that provide information about an object's X-ray absorption, refraction and...
We have developed two phase-retrieval techniques for analyser-based phase contrast imaging that provide information about an object's X-ray absorption, refraction and scattering properties. The first requires rocking curves to be measured with and without the sample and improves upon existing techniques by accurately fitting the curves with Pearson type VII functions. The second employs an iterative approach using two simultaneously recorded images by exploiting the Laue crystal geometry. This technique provides a substantial reduction in X-ray dose and enables quantitative phase retrieval to be performed on images of moving objects.
Topics: Algorithms; Microscopy, Phase-Contrast; Radiographic Image Enhancement; Radiographic Image Interpretation, Computer-Assisted
PubMed: 20940891
DOI: 10.1364/OE.18.019994 -
The Malaysian Journal of Pathology Dec 2010Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with...
Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.
Topics: Dendritic Cells; Humans; Microscopy, Confocal; Microscopy, Phase-Contrast
PubMed: 21329180
DOI: No ID Found -
Journal of Biomedical Optics 2011Tomographic phase microscopy measures the 3-D refractive index distribution of cells and tissues by combining the information from a series of angle-dependent...
Tomographic phase microscopy measures the 3-D refractive index distribution of cells and tissues by combining the information from a series of angle-dependent interferometric phase images. In the original device, the frame rate was limited to 0.1 frames per second (fps) by the technique used to acquire phase images, preventing measurements of moving or rapidly changing samples. We describe an improved tomographic phase microscope in which phase images are acquired via a spatial fringe pattern demodulation method, enabling a full tomogram acquisition rate of 30 fps. In addition, in this system the refractive index is calculated by a diffraction tomography algorithm that accounts for the effects of diffraction in the 3-D reconstruction. We use the instrument to quantitatively monitor rapid changes in refractive index within defined subregions of cells due to exposure to acetic acid or changes in medium osmolarity.
Topics: Computer-Aided Design; Equipment Design; Equipment Failure Analysis; Image Enhancement; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Microscopy, Video; Reproducibility of Results; Sensitivity and Specificity; Tomography, Optical
PubMed: 21280892
DOI: 10.1117/1.3522506 -
PloS One 2017Cardiovascular diseases remain the number one death cause worldwide. Preclinical 4D flow phase contrast magnetic resonance imaging can provide substantial insights in...
PURPOSE
Cardiovascular diseases remain the number one death cause worldwide. Preclinical 4D flow phase contrast magnetic resonance imaging can provide substantial insights in the analysis of aortic pathophysiologies in various animal models. These insights may allow a better understanding of pathophysiologies, therapy monitoring, and can possibly be translated to humans. This study provides a framework to acquire the velocity field within the aortic arch. It analyses important flow values at different locations within the aortic arch. Imaging parameters with high temporal and spatial resolution are provided, that still allow combining this time-consuming method with other necessary imaging-protocols.
METHODS
A new setup was established where a prospectively gated 4D phase contrast sequence is combined with a highly sensitive cryogenic coil on a preclinical magnetic resonance scanner. The sequence was redesigned to maintain a close to steady state condition of the longitudinal magnetization and hence to overcome steady state artifacts. Imaging parameters were optimized to provide high spatial and temporal resolution. Pathline visualizations were generated from the acquired velocity data in order to display complex flow patterns.
RESULTS
Our setup allows data acquisition with at least two times the rate than that of previous publications based on Cartesian encoding, at an improved image quality. The "steady state" sequence reduces observed artifacts and provides uniform image intensity over the heart cycle. This made possible quantification of blood speed and wall shear stress (WSS) within the aorta and its branches. The highest velocities were observed in the ascending aorta with 137.5 ± 8 cm/s. Peak velocity values in the Brachiocephalic trunk were 57 ± 12 cm/s. Quantification showed that the peak flow occurs around 20 ms post R-wave in the ascending aorta. The highest mean axial wall shear stress was observed in the analysis plane between the left common carotid artery (LCCA) and the left subclavian artery. A stable image quality allows visualizing complex flow patterns by means of streamlines and for the first time, to the best of our knowledge, pathline visualizations from 4D flow MRI in mice.
CONCLUSION
The described setup allows analyzing pathophysiologies in mouse models of cardiovascular diseases in the aorta and its branches with better image quality and higher spatial and temporal resolution than previous Cartesian publications. Pathlines provide an advanced analysis of complex flow patterns in the murine aorta. An imaging protocol is provided that offers the possibility to acquire the aortic arch at sufficiently high resolution in less than one hour. This allows the combination of the flow assessment with other multifunctional imaging protocols.
Topics: Animals; Aorta, Thoracic; Blood Flow Velocity; Image Interpretation, Computer-Assisted; Magnetic Resonance Imaging; Mice; Microscopy, Phase-Contrast; Systole
PubMed: 29117252
DOI: 10.1371/journal.pone.0187596 -
Journal of Biomedical Optics Jul 2012We present a quadriwave lateral shearing interferometer used as a wavefront sensor and mounted on a commercial non-modified transmission white-light microscope as a...
We present a quadriwave lateral shearing interferometer used as a wavefront sensor and mounted on a commercial non-modified transmission white-light microscope as a quantitative phase imaging technique. The setup is designed to simultaneously make measurements with both quantitative transmission phase and fluorescence modes: phase enables enhanced contrasted visualization of the cell structure including intracellular organelles, while fluorescence allows a complete and precise identification of each component. After the characterization of the phase measurement reliability and sensitivity on calibrated samples, we use these two imaging modes to measure the characteristic optical path difference between subcellular elements (mitochondria, actin fibers, and vesicles) and cell medium, and demonstrate that phase-only information should be sufficient to identify some organelles without any labeling, like lysosomes. Proof of principle results show that the technique could be used either as a qualitative tool for the control of cells before an experiment, or for quantitative studies on morphology, behavior, and dynamics of cells or cellular components.
Topics: Animals; COS Cells; Chlorocebus aethiops; Computer-Aided Design; Equipment Design; Equipment Failure Analysis; Image Enhancement; Interferometry; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Pilot Projects; Reproducibility of Results; Sensitivity and Specificity; Subcellular Fractions
PubMed: 22894487
DOI: 10.1117/1.JBO.17.7.076004 -
Optics Express Jul 2013In spiral phase contrast (SPC) microscopy the edge-enhancement is typically independent of the helicity of the phase vortex filter. Here we show that for layered...
In spiral phase contrast (SPC) microscopy the edge-enhancement is typically independent of the helicity of the phase vortex filter. Here we show that for layered specimens containing screw-dislocations, as are e.g. present in mica or some crystallized organic substances, the intensity distribution in the filtered image acquires a dependence on the rotational direction of the filter. This allows one to map the distribution of phase singularities in the topography of the sample, by taking the intensity difference between two images recorded with opposite handedness. For the demonstration of this feature in a microscopy set-up, we encode the vortex filter as a binary off-axis hologram displayed on a spatial light modulator (SLM) placed in a Fourier plane. Using a binary grating, the diffraction efficiencies for the plus and minus first diffraction orders are equal, giving rise to two image waves which travel in different directions and are Fourier filtered with opposite helicity. The corresponding two images can be recorded simultaneously in two separate regions of the camera chip. This enables mapping of dislocations in the sample in a single camera exposure, as was demonstrated for various transparent samples.
Topics: Equipment Design; Equipment Failure Analysis; Holography; Image Enhancement; Microscopy, Phase-Contrast; Refractometry
PubMed: 23938479
DOI: 10.1364/OE.21.016282