利用定量相衬成像消除X射线同轴轮廓成像中散射的影响

在所有的相衬成像方法中，X射线同轴轮廓成像由于光路简单、不需要任何光学元件而备受关注.其缺点是无法直接消除散射的影响，从而限制了它在生物医学等领域中的应用.利用数字模拟方法研究了引入散射前后轮廓像的质量随样品到探测器距离的变化情况.模拟结果表明直接轮廓成像存在一个最佳成像距离，因而无法通过改变样品到探测器距离来减小散射的影响.利用定量相衬成像不受成像距离限制的特点，研究了远距离成像时散射的影响.结果表明，样品到探测器的距离增大到一个临界值时，散射对重构像的影响可降到一个极值，此时成像质量得到了明显改善. 关键词： 散射 X射线同轴轮廓成像 定量相衬成像     

Performance analysis of quantitative phase retrieval method in Zernike phase contrast X-ray microscopy

Since the invention of Zernike phase contrast method in 1930,it has been widely used in optical microscopy and more recently in X-ray microscopy.Considering the image contrast is a mixture of absorption and phase information,we recently have proposed and demonstrated a method for quantitative phase retrieval in Zernike phase contrast X-ray microscopy.In this contribution,we analyze the performance of this method at different photon energies.Intensity images of PMMA samples are simulated at 2.5 keV and 6.2 keV,respectively,and phase retrieval is performed using the proposed method.The results demonstrate that the proposed phase retrieval method is applicable over a wide energy range.For weakly absorbing features,the optimal photon energy is 2.5 keV,from the point of view of image contrast and accuracy of phase retrieval.On the other hand,in the case of strong absorption objects,a higher photon energy is preferred to reduce the error of phase retrieval.These results can be used as guidelines to perform quantitative phase retrieval in Zernike phase contrast X-ray microscopy with the proposed method.     

高对比度目标的电磁逆散射超分辨成像

提出了一种适用于高对比度目标的超分辨成像方法,通过结合对比度源反演方法与基于轨道角动量的超分辨技术,实现对高对比度目标的超分辨成像.首先采用基于轨道角动量的成像方法求解出对比度函数,将其作为对比度源反演方法的迭代初值,虽然初值结果与实际目标相差较大,但是由于初值中已经包含了关于目标的倏逝波信息,再利用这个初值开始迭代便可以得到超分辨重建结果,这种方法具有一定的抗噪声能力.本文研究表明,为了实现超分辨成像,一方面需要将目标对应的倏逝波信息转化到测量数据中,另一方面还要保证成像算法能够充分利用这些信息.本文所引申出的关于超分辨信息的概念对于逆散射超分辨成像的研究具有一定的借鉴意义.     

Simple phase extraction in x-ray differential phase contrast imaging

A fast and simple method to extract phase-contrast images from interferograms is proposed, and its effectiveness is demonstrated through simulation and experiment. For x-ray differential phase contrast imaging, a strong attenuation signal acts as an overwhelming background intensity that obscures the weak phase signal so that no obvious phase-gradient information is detectable in the raw image. By subtracting one interferogram from another, chosen at particular intervals,the phase signal can be isolated and magnified.     

Leucine pools in Escherichia coli biofilm discovered by Raman imaging

In structured communities of bacteria known as biofilms, a variety of biomolecules have been shown to play a unique role as signals and/or regulators in biofilm formation. Here, we report that high levels of the amino acid leucine (leucine pool) were detected, for the first time, within microcolonies in a 30‐h‐old Escherichia coli biofilm by Raman imaging. Localization of leucine revealed by multifrequency Raman images indicates leucine accumulation during the early stage of the E. coli biofilm formation, which may have resulted from physiological environment‐specific metabolic adaptation. We demonstrate that our label‐free Raman imaging method provides a useful platform for directly identifying still unknown natural products produced in biofilms as well as for visualizing heterogeneous distributions of biofilm constituents in situ. Copyright © 2011 John Wiley & Sons, Ltd.     

Programmable aperture microscopy: A computational method for multi-modal phase contrast and light field imaging

We demonstrate a simple and cost-effective programmable aperture microscope to realize multi-modal computational imaging by integrating a programmable liquid crystal display (LCD) into a conventional wide-field microscope. The LCD selectively modulates the light distribution at the rear aperture of the microscope objective, allowing numerous imaging modalities, such as bright field, dark field, differential phase contrast, quantitative phase imaging, multi-perspective imaging, and full resolution light field imaging to be achieved and switched rapidly in the same setup, without requiring specialized hardwares and any moving parts. We experimentally demonstrate the success of our method by imaging unstained cheek cells, profiling microlens array, and changing perspective views of thick biological specimens. The post-exposure refocusing of a butterfly mouthpart and RFP-labeled dicot stem cross-section is also presented to demonstrate the full resolution light field imaging capability of our system for both translucent and fluorescent specimens.     

基于光强传输方程相位成像的宽场相干反斯托克斯拉曼散射显微背景抑制

相干反斯托克斯拉曼散射(CARS)显微能够对样品的特殊化学组分进行选择性成像,无需荧光标记,在生物医学领域被广泛应用.然而,传统的CARS图像往往存在非共振背景信号.本文将基于光强传输方程的单光束相位成像技术用于CARS显微成像,来抑制CARS的非共振背景信号.该方法通过记录样品在三个相邻平面上的CARS图像,然后利用光强传输方程获取CARS光场的相位分布,最后利用共振CARS信号和非共振背景信号在相位上的差异,实现了对背景噪声的抑制.该方法无需参考光,通过三次测量可完成CARS的背景噪声抑制,具有良好的应用前景.     

Improvement and error analysis of quantitative information extraction in diffraction-enhanced imaging

Diffraction-enhanced imaging （DEI） is a powerful phase-sensitive technique that provides higher spatial resolution and supercontrast of weakly absorbing objects than conventional radiography. It derives contrast from the X-ray absorption, refraction, and ultra-small-angle X-ray scattering （USAXS） properties of an object. The separation of different-contrast contributions from images is an important issue for the potential application of DEI. In this paper, an improved DEI （IDEI） method is proposed based on the Gaussian curve fitting of the rocking curve （RC）. Utilizing only three input images, the IDEI method can accurately separate the absorption, refraction, and USAXS contrasts produced by the object. The IDEI method can therefore be viewed as an improvement to the extended DEI （EDEI） method. In contrast, the IDEI method can circumvent the limitations of the EDEI method well since it does not impose a Taylor approximation on the RC. Additionally, analysis of the IDEI model errors is performed to further investigate the factors that lead to the image artifacts, and finally validation studies are conducted using computer simulation and synchrotron experimental data.     

Non‐invasive time‐course imaging of apoptotic cells by confocal Raman micro‐spectroscopy

Confocal Raman micro‐spectroscopy (CRMS) was used to measure time‐course spectral images of live cells undergoing apoptosis without using molecular labels or other invasive procedures. Human breast cancer cells (MDA‐MB‐231) were exposed to 300 µM etoposide to induce apoptosis, and Raman spectral images were acquired from the same cells at 2‐h intervals over a period of 6 h. The purpose‐built inverted confocal Raman micro‐spectrometer integrated an environmental enclosure and wide‐field fluorescence imaging. These key instrumental elements allowed the cells to be maintained under sterile physiological conditions (37 °C, 5% CO₂) and enabled viability and apoptosis assays to be carried out on the cells at the end of CRMS measurements. The time‐course spectral images corresponding to DNA Raman bands indicated an increase in signal intensity in apoptotic cells, which was attributed to DNA condensation. The Raman spectral images of lipids indicated a high accumulation of membrane phospholipids and highly unsaturated non‐membrane lipids in apoptotic cells. This study demonstrates the potential of CRMS for label‐free time‐course imaging of individual live cells. This technique may become a useful tool for in vitro toxicological studies and testing of new pharmaceuticals, as well as other time‐dependent cellular processes, such as cell differentiation, cell cycle and cell–cell interactions. Copyright © 2010 John Wiley & Sons, Ltd.     

相干X射线衍射成像技术及在材料学和生物学中的应用

无损获取高分辨率、高衬度微纳米材料结构图像,并能够原位、定量分析是X射线成像技术发展方向之一.最新发展起来的相干X射线衍射成像技术,也称为无透镜成像技术,为实现这一目标提供了可能.本文介绍了相干X射线衍射成像技术的成像原理和发展过程,以及在材料学和生物学中的一些典型应用和最新进展,例如掺杂铋元素在硅晶体中的分布成像,GaN量子点壳层结构的三维高分辨成像,染色大肠杆菌的二维成像,鱼骨的二维成像及矿化机理研究,单个未染色疱疹病毒的二维高衬度成像,未染色酵母菌细胞的三维高分辨成像及原位定量分析.最后对相干X射线衍射成像技术的发展方向做了展望.随着X射线自由电子激光的应用和冷冻技术与相干X射线衍射成像技术的结合,相干X射线衍射成像技术将得到快速的发展和广泛的应用.     

A new method for information retrieval in two-dimensional grating-based X-ray phase contrast imaging

Grating-based X-ray phase contrast imaging has been demonstrated to be an extremely powerful phase-sensitive imaging technique.By using two-dimensional(2D) gratings,the observable contrast is extended to two refraction directions.Recently,we have developed a novel reverse-projection(RP) method,which is capable of retrieving the object information efficiently with one-dimensional(1D) grating-based phase contrast imaging.In this contribution,we present its extension to the 2D grating-based X-ray phase contrast imaging,named the two-dimensional reverseprojection(2D-RP) method,for information retrieval.The method takes into account the nonlinear contributions of two refraction directions and allows the retrieval of the absorption,the horizontal and the vertical refraction images.The obtained information can be used for the reconstruction of the three-dimensional phase gradient field,and for an improved phase map retrieval and reconstruction.Numerical experiments are carried out,and the results confirm the validity of the 2D-RP method.     

A protocol for searching the most probable phase‐retrieved maps in coherent X‐ray diffraction imaging by exploiting the relationship between convergence of the retrieved phase and success of calculation

Coherent X‐ray diffraction imaging (CXDI) is a technique for visualizing the structures of non‐crystalline particles with size in the submicrometer to micrometer range in material sciences and biology. In the structural analysis of CXDI, the electron density map of a specimen particle projected along the direction of the incident X‐rays can be reconstructed only from the diffraction pattern by using phase‐retrieval (PR) algorithms. However, in practice, the reconstruction, relying entirely on the computational procedure, sometimes fails because diffraction patterns miss the data in small‐angle regions owing to the beam stop and saturation of the detector pixels, and are modified by Poisson noise in X‐ray detection. To date, X‐ray free‐electron lasers have allowed us to collect a large number of diffraction patterns within a short period of time. Therefore, the reconstruction of correct electron density maps is the bottleneck for efficiently conducting structure analyses of non‐crystalline particles. To automatically address the correctness of retrieved electron density maps, a data analysis protocol to extract the most probable electron density maps from a set of maps retrieved from 1000 different random seeds for a single diffraction pattern is proposed. Through monitoring the variations of the phase values during PR calculations, the tendency for the PR calculations to succeed when the retrieved phase sets converged on a certain value was found. On the other hand, if the phase set was in persistent variation, the PR calculation tended to fail to yield the correct electron density map. To quantify this tendency, here a figure of merit for the variation of the phase values during PR calculation is introduced. In addition, a PR protocol to evaluate the similarity between a map of the highest figure of merit and other independently reconstructed maps is proposed. The protocol is implemented and practically examined in the structure analyses for diffraction patterns from aggregates of gold colloidal particles. Furthermore, the feasibility of the protocol in the structure analysis of organelles from biological cells is examined.     

Quantitative,comparable coherent anti‐Stokes Raman scattering (CARS) spectroscopy: correcting errors in phase retrieval

Coherent anti‐Stokes Raman scattering (CARS) microspectroscopy has demonstrated significant potential for biological and materials imaging. To date, however, the primary mechanism of disseminating CARS spectroscopic information is through pseudocolor imagery, which explicitly neglects a vast majority of the hyperspectral data. Furthermore, current paradigms in CARS spectral processing do not lend themselves to quantitative sample‐to‐sample comparability. The primary limitation stems from the need to accurately measure the so‐called nonresonant background (NRB) that is used to extract the chemically sensitive Raman information from the raw spectra. Measurement of the NRB on a pixel‐by‐pixel basis is a nontrivial task; thus, surrogate NRB from glass or water is typically utilized, resulting in error between the actual and estimated amplitude and phase. In this paper, we present a new methodology for extracting the Raman spectral features that significantly suppresses these errors through phase detrending and scaling. Classic methods of error correction, such as baseline detrending, are demonstrated to be inaccurate and to simply mask the underlying errors. The theoretical justification is presented by re‐developing the theory of phase retrieval via the Kramers–Kronig relation, and we demonstrate that these results are also applicable to maximum entropy method‐based phase retrieval. This new error‐correction approach is experimentally applied to glycerol spectra and tissue images, demonstrating marked consistency between spectra obtained using different NRB estimates and between spectra obtained on different instruments. Additionally, in order to facilitate implementation of these approaches, we have made many of the tools described herein available free for download. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.     

弱散射体产生的菲涅耳极深区散斑场相位涡旋演化的实验研究

实验提取了弱散射体产生的菲涅耳极深区的散斑场.发现当散射距离一定时,弱散射体光场的相位分布特征随散射体的粗糙度的变化而变化;对于某一弱散射体,相位分布特征随散射距离的变化而变化;当弱散射体的粗糙度大到一定程度时,才产生相位涡旋现象;散射体表面上存在相位涡旋;弱散射体产生的相位涡旋的密度随散射屏粗糙度的增大而增大,还随散射距离的增大而增大.研究结果对于认识弱散射体的相位及相位涡旋分布特征随粗糙度和散射距离的演化具有重要意义,而且对于认识散斑场随散射距离的演化有一定的帮助.     

Dynamics of radiation scattering by particles of condensed phase in quasicontinuous laser irradiation of metals

Radiation scattering by particles of condensed phase in an ablation plasma plume has been experimentally studied during quasicontinuous laser irradiation (λ = 1.06 μm, q = 0.1–9 MW/cm², τ ∼ 1.5 msec) of duraluminum D16T, aluminum A99, and bismuth. The particle size distribution and the nature of their dispersal during irradiation was studied in scattered light (λ = 0.69 μm) from individual particles that could be visually observed on photographs. It was found that under the pressure developed in the plume, large particles ejected from the irradiated zone can move backward and return to the target (D16T). The plume (Bi) becomes brighter due to ablation of particles in the path of the laser beam. The directional scattering coefficients for scattering from the local zone on the axis of the plume, measured during the laser pulse, were used to study the relationship between the dynamics of entry of condensed phase into the plume, shielding of the target by the particles, and brightening of the plume under the action of the incident laser radiation. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 2, pp. 210–219, March–April, 2006.     

Synchrotron X‐ray phase contrast imaging of leaf venation in soybean (Glycine max) after exclusion of solar UV (280–400 nm) radiation

The hydraulic efficiency of a leaf depends on its vascular structure as this is responsible for transport activities. To investigate the effect of exclusion of UVAB and UVB radiation from the solar spectrum on the micro‐structure of leaves of soybean (Glycine max, variety JS‐335), a field experiment was conducted using synchrotron‐based phase contrast imaging (PCI). Plants were grown in specially designed UV exclusion chambers, and wrapped with filters that excluded UVB (280–315 nm) or UVAB (280–400 nm), or transmitted all the ambient solar UV (280–400 nm) radiation (filter control). Qualitative observation of high‐resolution X‐ray PCI images obtained at 10 keV has shown the differences in major and minor vein structures of the leaves. The mid‐rib width of the middle leaflet of third trifoliate leaves, for all treatments, were obtained using quantitative image analysis. The width of the mid‐rib of the middle leaflet of third trifoliate leaves of UVB excluded plants was found to be more compared to leaves of filter control plants, which are exposed to ambient UV. The mid‐rib or the main conducting vein transports water and sugars to the whole plant; therefore, mid‐rib enhancement by the exclusion of solar UV radiation possibly implies enhancement in the leaf area which in turn causes an increased rate of photosynthesis.     

Low‐noise,vibrational phase‐sensitive chemical imaging by balanced detection RIKE

We perform a back‐to‐back comparison between two nonlinear vibrational imaging techniques: stimulated Raman scattering (SRS) and balanced detection Raman‐induced Kerr effect (BD‐RIKE). Using a compact fiber‐based laser system for generation of pump and Stokes signals, we image polymer beads as well as human hepatocytes under the same experimental conditions. We show that BD‐RIKE, despite the slightly lower signal levels, consistently offers an improved signal‐to‐noise ratio with respect to SRS, resulting in significantly higher image quality. Importantly, we observe that such quality is not affected by the static birefringence of the sample, which makes BD‐RIKE a robust and attractive alternative to SRS. We also highlight a unique advantage of the technique, which is its capability to easily access both the real and imaginary parts of the nonlinear susceptibility, thus allowing for vibrational phase imaging. The phase information can be readily obtained from BD‐RIKE with minimal experimental effort and provides an additional chemical selectivity channel for coherent Raman microscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Orientational diffusivities measured by Rayleigh scattering in a lyotropic calamitic nematic () liquid crystal phase: the backflow problem revisited

Using a light-beating technique we have measured the damping time of thermal fluctuations of the nematic director for the so called cylindrical or calamitic nematic (N_C) phase of the lyotropic system K-laurate/decanol/. By varying the scattering angle in suitable geometries, we have been able to estimate the orientational diffusivities associated to the three pure deformations of splay, twist and bend. A former measurement made in the disk-like N_D phase of the same system yielded a large deviation between the splay and twist diffusivities. The effect was then attributed to induced flows, or backflow, which could be responsible for the reduction of the splay viscosity. In fact, this is the analogous effect, for disks, to the one recognized since long time ago arriving for rod-like molecules in a classical nematic, though in this case it is associated with bend deformations. The analogy comes about thanks to the interchange of the role played by disks and cylinders for, respectively, splay and bend fluctuations.The measurements reported here provide a new test on the applicability of the backflow model to a nematic system composed of micelles, that is, aggregates made of amphiphilic (surfactant) molecules, in its cylindrical-like variant, i.e. the N_C phase. In addition, the comparative study made here with the previous results existing in the literature for the N_D phase, allows us to conjecture on structural issues concerning lyotropic nematics. Received: 29 April 1998 / Revised: 19 August 1998 / Accepted: 31 August 1998