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Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (<1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy. Until now, X-ray microscopes operating in the "water window" energy range between carbon and oxygen k-shell absorption edges have produced outstanding 3D images of cryo-preserved cells. The relatively low X-ray energy (<540 eV) of the water window imposes two important limitations: limited penetration (<10 μm) not suitable for imaging larger cells or tissues, and small depth of focus (DoF) for high resolution 3D imaging (e.g., ~1 μm DoF for 20 nm resolution). An X-ray microscope operating at intermediate energy around 2.5 keV using Zernike phase contrast can overcome the above limitations and reduces radiation dose to the specimen. Using a hydrated model cell with an average chemical composition reported in literature, we calculated the image contrast and the radiation dose for absorption and Zernike phase contrast, respectively. The results show that an X-ray microscope operating at ~2.5 keV using Zernike phase contrast offers substantial advantages in terms of specimen size, radiation dose and depth-of-focus., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples.

X-ray grating interferometry provides substantially increased contrast over conventional absorption-based imaging methods, and therefore new and complementary information. Compared with other phase-contrast imaging techniques, x-ray grating interferometry can overcome some of the problems that have impaired the applications of x-ray phase-contrast radiography and phase tomography. Recently, special attention has been paid to the development of quantitative phase retrieval methods, which is mandatory to perform x-ray phase tomography, to achieve material identification, to differentiate distinct tissues, etc. Typically, the phase-stepping approach has been utilized for phase retrieval in grating interferometry. This method requires a grating scanning and acquisition of multiple radiographic projections, and therefore is disadvantageous in terms of imaging speed and radiation damage. Here we present an innovative, highly sensitive approach, dubbed ‘reverse projection’ (RP), for quantitative phase retrieval. Compared with the phase-stepping approach, the present RP method abandons grating scanning completely, and thus is advantageous due to its much higher efficiency and the reduced radiation dose, without the degradation of reconstruction quality. This review presents a detailed explanation of the principle of the RP method. Both radiography and phase tomography experiments are performed to validate the RP method. We believe that this new technique will find widespread applications in biomedical imaging and in vivo studies. [ABSTRACT FROM AUTHOR]

Impressive developments in X-ray imaging are associated with X-ray phase contrast computed tomography based on grating interferometry, a technique that provides increased contrast compared with conventional absorption-based imaging. A new 'single-step' method capable of separating phase information from other contributions has been recently proposed. This approach not only simplifies data-acquisition procedures, but, compared with the existing phase step approach, significantly reduces the dose delivered to a sample. However, the image reconstruction procedure is more demanding than for traditional methods and new algorithms have to be developed to take advantage of the 'single-step' method. In the work discussed in this paper, a fast iterative image reconstruction method named OSEM (ordered subsets expectation maximization) was applied to experimental data to evaluate its performance and range of applicability. The OSEM algorithm with different subsets was also characterized by comparison of reconstruction image quality and convergence speed. Computer simulations and experimental results confirm the reliability of this new algorithm for phase-contrast computed tomography applications. Compared with the traditional filtered back projection algorithm, in particular in the presence of a noisy acquisition, it furnishes better images at a higher spatial resolution and with lower noise. We emphasize that the method is highly compatible with future X-ray phase contrast imaging clinical applications. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the use of transmission hard X-ray nano-computed-tomography (nano-CT) for characterization of the pore structure and drug distribution in poly (lactic-co-glycolic acid) (PLGA) microspheres encapsulating bovine serum albumin and to study the correlation between drug distribution and burst release. The PLGA microspheres were fabricated using a double-emulsion method. The results of pore structure analysis accessed with nano-CT were compared with those acquired by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Surface pore interconnectivity and surface protein interconnectivity were obtained using combined nano-CT and pixel analysis. The correlation between surface protein interconnectivity with the initial burst release across various tested formulations was also analyzed. The size, shape, and distribution of the pores and protein could be clearly observed in the whole microsphere using nano-CT, whereas only the sectional information was observed using SEM or CLSM. Interconnected pores and surface connected pores could be clearly distinguished in nano-CT, which enables the quantitative analysis of surface pore interconnectivity and surface protein interconnectivity. The surface protein interconnectivity in different formulations correlated well with the burst release at 5–10 h. Nano-CT provided a nondestructive, high-resolution, and three-dimensional analysis method to characterize the porous microsphere. [ABSTRACT FROM AUTHOR]

Abstract: X-ray phase contrast imaging has been demonstrated to have an improved contrast over conventional absorption imaging for those weakly absorbing objects. However, most of the hard X-ray phase-sensitive imaging has so far been impractical with laboratory available X-ray sources. Grating-based phase imaging approach has the prominent advantage that polychromatic laboratory X-ray generators can be efficiently used in a Talbot–Lau configuration. Through numerical simulations, we demonstrate here the efficient use of polychromatic X-ray laboratory sources for differential phase contrast imaging. The presented results explain why in recently reported experiments, polychromatic X-ray tubes could be efficiently used in a Talbot–Lau interferometer. Furthermore, the results indicate that the fractional Talbot distance is not limited by the polychromaticity of the X-ray source. Since the sensitivity of phase measurements is proportional to the fractional Talbot distance, the image quality for phase measurements can be further improved. Finally, the potential optimizations of the imaging system are discussed from an application perspective, taking into consideration both available X-ray flux and compactness of the system. [Copyright &y& Elsevier]

Abstract: A new diffraction-enhanced imaging (DEI) set-up was designed and constructed at Beijing Synchrotron Radiation Facility. The size of imaging beam cross section is the same as that of incident “white” X-ray beam in the new set-up. While in the original set-up, the size is always limited in one direction. Thus, larger samples can be imaging in a single exposure without need for scanning of the sample and detector in the new set-up. Choosing a fly as sample, DEI and DEI-computed-tomography (CT) experiments were carried out using the new set-up. Experimental results show that the spatial resolution of the CT image is in the tens of microns. [Copyright &y& Elsevier]

Abstract: The 4W1A beamline at the Beijing Synchrotron Radiation Facility (BSRF) had been dedicated to researches in the field of X-ray phase contrast imaging. In both available layouts, e.g., in-line imaging and diffraction enhanced imaging, a spatial resolution better than 10μm has been achieved and an X-ray phase contrast CT system has been installed and tested on the beamline. With or without analyzer, it can work either in in-line or the diffraction enhanced mode. [Copyright &y& Elsevier]