基于同步辐射X射线的多尺度生物成像研究

发布时间：2018-01-16 09:10

  本文关键词：基于同步辐射X射线的多尺度生物成像研究　出处：《山东大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 生物成像 低辐射剂量 多尺度 同步辐射X射线 断层扫描成像

【摘要】：对生物显微结构的认识横跨多个尺度,包括生物个体、组织到细胞等。实现多尺度的生物系统的成像对于生物科学的发展有重要的意义。x射线因其波长短和穿透能力强可以实现多尺度生物样品的无损、三维、高分辨成像,弥补了光学显微镜和电子显微镜在生物成像中存在的不足。随着第三代同步辐射X射线光源和光学器件制作工艺的发展,X射线成像技术逐渐成为生物结构研究的重要工具。但是,在X射线成像方法及应用中仍然存在一定的问题,包括生物样品在成像过程中的辐射损伤问题,三维重建算法的精确程度,以及三维重建图像的后期图像处理等。针对X射线成像中存在的科学问题,本文开展了基于同步辐射X射线的多尺度生物成像方法学探索,并在此基础上对细胞及昆虫等生物样品的三维高分辨成像进行了应用研究。具体内容包括：(1)基于同步辐射X射线的三维、高分辨、低剂量细胞成像和细胞内纳米材料的双能衬度X射线成像方法及应用研究。结合扫描透射X射线成像技术(Scanning Transmission X-ray Microscopy, STXM)和等斜率层析算法(Equally Sloped Tomography, EST)实现对[Gd@C82(OH)22]n溶液中培养的贴壁巨噬细胞的三维高分辨成像。在Gd的M2吸收边前(能量1186eV)和吸收边上(能量1189eV)按等斜率方式分别采集了两套数据(每套包含46个投影,角度分布±79.4°)。通过EST重建获得巨噬细胞在50 nm像素分辨率下的三维显微结构。观察到细胞的特征性表面形貌,例如扁平的三维结构,伸出的触手；识别出细胞核、溶酶体等不同的细胞器。通过在实验过程中采用扫描成像模式和三维重建过程中采用新的图像重建算法,使得生物样品的辐射剂量大幅度降低到4.43×106 Gy。根据已有研究,该辐射剂量不会引起细胞结构的改变；同时,通过对比实验前后细胞在0°的投影,进一步证实了结构没有发生变化。等斜率迭代算法的应用有效地解决了实验数据有限,以及采集过程中高角度投影缺失的问题,同时提高了三维图像重建的准确度,有利于得到细胞的精确三维结构。系统研究了双能衬度x射线成像技术,根据x射线的吸收在特定元素吸收边能量前后发生突变的特性,实现了巨噬细胞内纳米材料[Gd@C82(OH)22]n的三维定量成像。分析对比了双能衬度成像方法中两种计算三维元素分布的途径：第一种,首先计算所有角度的二维元素分布,然后经旋转轴校正和强度归一化后进行三维重建,得到三维元素分布；第二种,先分别对两个能量的数据进行旋转轴校准和强度的校正,然后进行三维重建,得到两个能量对应的三维图像,随后配准两个三维图像,计算二者差异得到三维元素分布。对比发现第二种方法可以得到更好的图像效果,有更高的信噪比,同时第二种更简单易于操作。基于第二种方法得到的结果,分析了巨噬细胞内Gd元素(即[Gd@C82(OH)22]n)的三维空间分布,与同一细胞的x射线荧光成像(X-Ray Fluorescence, XRF)相比具有更好的图像质量和分辨能力。实验结果显示[Gd@C82(OH)22]n发生聚集并被巨噬细胞大量吞入,主要分布在溶酶体内,但并未进入细胞核。其它相同条件培养得到的细胞也证实了纳米材料在巨噬细胞内具有类似的空间分布。对巨噬细胞的形貌、细胞内纳米材料的空间分布以及生理分析将有助于理解[Gd@C82(OH)22]n这种潜在的纳米药物在体内和体外均显示高效低毒的抗肿瘤效果。同时,结合EST算法和STXM对贴壁生长的Ag标定的淋巴细胞进行了三维成像研究。在1060 eV采集了37个等斜率投影,角度分布为-63.44°到69.44°,聚焦光斑直径50 nm,扫描步进50 nm。通过EST重建得到细胞的三维表面形貌,尺寸-20x17×5μm3,但内部结构信息不易分辨。主要原因是细胞的厚度超出了波带片的焦深范围,造成图像的模糊,无法识别细胞内的精细结构；1060 eV的软x射线对于样品的透过率很低,造成高密度或者较厚的区域无法分辨。另外,由于标定的Ag颗粒尺寸远小于成像分辨率,目前无法根据吸收衬度的差异实现对单个颗粒成像识别。通过对成像参数例如能量、样品厚度、波带片焦深等对三维成像质量影响的分析,增进了对于细胞三维成像方法的理解,对生物样品成像实验提供了指导。(2)活体昆虫的同步辐射X射线相位衬度成像研究。研究包括两个方面,首先通过对干燥黄粉虫吸收衬度成像,验证EST算法的高效和准确性；然后对活体黄粉虫进行同轴相位衬度X射线成像。首先,对干燥的黄粉虫进行同步辐射X射线吸收衬度显微成像,在12 KeV采集了320个等斜率投影和600个等角度投影,单张曝光55 ms,分别用EST算法和滤波反投影算法(Filtered Back Projection algorithm, FBP)进行图像重建。通过改变两种算法在重建过程中所用的投影数目,系统地对比了两种重建算法的三维重建效果。信噪比、衬度和噪声功率谱等图像参数都显示采用EST算法时320个投影得到的图像重建效果远远好于采用FBP算法时600个投影的重建效果。即使用180个投影,采用EST算法也可以得到显微结构可识别的三维图像。对比结果显示EST算法可以在保持分辨率的情况下减少三分之二的投影数目。投影数目的减少可以有效降低生物样品的辐射剂量。根据EST算法的重建图像分析了干燥黄粉虫的三维表面形貌和内部显微结构,如肌肉组织等。利用同步辐射X射线,对冷冻麻醉固定活体黄粉虫开展了同轴相位衬度成像,定性和定量地分析昆虫在活体状态的内部显微结构。通过实验设备的改进,如快速成像探测器的使用和样品前快门的使用,并结合EST中等斜率数据采集模式极大地减少了投影数目,从而降低了辐射剂量,实验过程中采集320个投影的总曝光时间为1.92s,辐射剂量为1.2 Gy,该剂量远远小于生物样品的致死剂量,实验后黄粉虫仍然存活。对实验数据分别进行了相位恢复前和相位恢复后的三维重建,得到9μm像素分辨率的三维结构信息。对未做相位恢复的三维图像重建,由于菲涅耳衍射效应造成的边缘增强效果,可以观测到内部气管及微气管等组织；而对于采用phase-attenuation-duality Born algorithm (PAD-BA)方法相位恢复后重建的图像,可根据三维折射率的空间分布进行定量的结构分析。本论文中采用的实验方法不仅可得到高分辨三维重建图像,同时还可以极大地减少样品的辐射剂量,在生物成像尤其是活体成像中会有重要的应用。(3)节肢动物的同步辐射X射线相位衬度成像研究。应用同步辐射X射线同轴相位衬度成像技术对毫米尺寸的节肢动物开展了无损三维结构研究。在12KeV能量下采集了干燥蚯蚓头部的450个等角度投影,单张曝光时间6s,利用FBP算法对未做相位恢复的投影和已做相位恢复的投影,分别进行三维重建,定性和定量地研究了蚯蚓的外部形貌和内部显微结构。根据不同组织的特异性结构和密度的差异,识别出来不同的内部显微组织,包括肌肉组织、血液循环系统、神经系统和消化系统。相对于常用的组织切片方法中对样品进行切片染色处理,然后用光学显微镜或电子显微镜观测,该种简易的同轴相位衬度成像方法可快速无损地得到样品的外部形貌和内部显微结构。因此,实验结果充分证明了相位衬度X射线在生物显微结构研究中的准确性和高效性,以及在研究低密度材料中的应用潜力。(4)显微CT图像中环形伪影的校正算法研究。基于正弦图处理的环形伪影校正方法可以在保持图像真实性的同时有效去除CT图像中的环形伪影。本论文提出并证实了一种基于正弦图处理的环形伪影校正新方法。和传统的正弦图处理的方法不同,本方法首先在正弦图中定位出线状伪影,然后在原始投影中把选出的线状伪影作为点噪音通过滤波去除。数值模拟和干燥黄粉虫的同步辐射X射线显微CT实验数据都显示环形伪影可以有效的去除而不会引起结构信息的丢失,证实了该方法的可行性和有效性。和常用正弦图方法的对比显示本文方法可以得到更好的校正效果。本文提出了一个环形伪影校正的新视角,通过结合更高效的线状伪影识别方法和点噪音去除算法,可以得到更好的环形伪影校正效果。
[Abstract]:Understanding of biological microstructure across multiple scales, including individual organisms, tissues to cells. The imaging of biological systems with multi scale for the development of biology science has important significance of.X rays because of its short wave length and strong penetration ability can achieve multi-scale biological samples without loss, three-dimensional, high resolution imaging, make up the shortcomings of optical microscope and electron microscope in biological imaging. With the development of fabrication of third generation synchrotron radiation X ray source and optical devices, X ray imaging technology has become an important tool for studying biological structures. However, there are still some problems in the X ray imaging method and application, including the damage of biological samples radiation in the imaging process, accurate 3D reconstruction algorithm, image reconstruction and image processing. According to the late science problems in X ray imaging In this paper, carry out multi-scale biological imaging method based on synchrotron radiation X ray exploration, and on the basis of 3D cell and insect biological samples such as high resolution imaging was studied. The specific contents include: (1) three dimensional, synchrotron radiation X ray based on high resolution, low doses of cells and cell imaging nano materials can double contrast X ray imaging method and application research. The combination of scanning transmission X-ray imaging technique X (Scanning Transmission X-ray Microscopy, STXM) and slope (Equally Sloped tomography algorithm Tomography, EST) the [Gd@C82 (OH) three adherent macrophages cultured in 22]n solution in high resolution imaging. Gd M2 (energy 1186eV) before the absorption edge and the absorption edge (energy 1189eV) by slope method were collected two sets of data (each containing 46 projection angle distribution, plus or minus 79.4 degrees) by EST reconstruction to obtain huge. The 3D microstructure of macrophages in the 50 nm pixel resolution. The observed characteristics of cell surface morphology, three-dimensional structure such as a flat, extending tentacles; identify nuclei, lysosomes in different organelles. By using the new image reconstruction algorithm using scanning imaging mode and three dimensional reconstruction during the experiment process so, the radiation dose of biological samples greatly reduced to 4.43 * 106 Gy. according to the previous studies, the radiation dose does not cause the change of cell structure; at the same time, through the contrast experiment before and after the projection cells in 0 degrees, further confirmed that the structure has not changed. The application of slope iterative algorithm can effectively solve the limited experimental data, the lack of high angle projection and acquisition process, and improve the accuracy of 3D image reconstruction, to obtain accurate 3D cell structure is investigated. Double contrast X ray imaging technology, according to X ray absorption and absorption edge energy in a particular element mutation characteristics, to achieve the [Gd@C82 in macrophages of nano materials (OH) three-dimensional quantitative imaging of 22]n. The comparative analysis of the two to two calculation approach of 3D element distribution contrast imaging method: first, first calculate the distribution of two-dimensional elements at all angles, and then reconstructed by the axis of rotation correction and normalization, three-dimensional element distribution; second, rotation axis calibration and correction for the strength of the first of two energy data, and then three-dimensional reconstruction, three-dimensional images of the two energy corresponding to the subsequent registration two a three-dimensional image, calculate the difference between the two three-dimensional element distribution. It is found that the second methods can get better image quality, higher signal-to-noise ratio, while second more easy In operation. The results of the second methods based on the analysis of the Gd element in macrophages ([Gd@C82 (OH) 22]n) distribution in three-dimensional space, X ray fluorescence imaging with the same cell (X-Ray Fluorescence, XRF) compared with better image quality and resolution. The experimental results show that [Gd@C82 (OH) 22]n aggregation and a large number of swallowed by macrophages, mainly in lysosomes, but did not enter the nucleus. Other conditions the same cultured cells also confirmed that nano materials have similar spatial distributions in macrophages. Morphology of macrophages, the spatial distribution of nano materials within the cell and physiological analysis will help [Gd@C82 (OH) 22]n nanoparticles showed the potential of high efficiency and low toxicity antitumor effect in vivo and in vitro. At the same time, the three combined with EST algorithm and STXM calibration of adherent Ag lymphocytes Study on dimensional imaging. In 1060 eV collected 37 isogradient projection angle distribution to 69.44 degrees -63.44 degrees, the focal spot diameter of 50 nm, 50 nm. EST scanning step through the reconstructed 3D surface topography of the cell, size of -20x17 * 5 m3, but the internal structure information is not easy to distinguish. The main reason is the cell thickness beyond the focal depth of plate, resulting in image blurring, the fine structure of cells is not recognized; 1060 eV soft X ray transmittance for the samples is very low, resulting in high density or thick regions can not be resolved. In addition, due to the particle size of Ag calibration is far less than the current imaging resolution. Not according to the absorption contrast differences of single particle image recognition. Based on imaging parameters such as energy, the thickness of the sample, analysis of deep influence on the quality of 3D imaging plate coke, enhance the cell for three-dimensional imaging method of understanding. The imaging experiment of biological samples provides guidance. (2) of the imaging study of synchrotron radiation X ray phase contrast in insects. Study includes two aspects, first through the absorption contrast imaging of dry Tenebrio molitor, to verify the EST algorithm with high efficiency and accuracy; and in vivo of Tenebrio molitor phase contrast X ray imaging. First of all, for the absorption contrast imaging synchrotron radiation X ray of dried Tenebrio molitor, in 12 KeV collected 320 and 600 angles of slope projection and projection, single exposure of 55 ms, respectively by EST algorithm and filter back projection algorithm (Filtered Back Projection algorithm, FBP) for image reconstruction. By changing the number of two kinds of projection algorithm used in the reconstruction process, the system compares the effects of two kinds of 3D reconstruction algorithm. The SNR, contrast and noise power spectrum of the image parameters are displayed when using EST algorithm 320 projection Image reconstruction effect is far better than the algorithm using FBP 600 projection reconstruction effect. Using 180 projection, using EST algorithm can obtain three-dimensional images of microstructure can be identified. The comparison results show that the EST algorithm can reduce the number of 2/3 projection while maintaining the resolution of the case. The number of projections can effectively reduce the number of the radiation dose of biological samples. According to the reconstructed image EST algorithm is analyzed. The 3D surface topography of Tenebrio molitor and internal microstructure, such as muscle tissue. The use of synchrotron radiation X ray, the refrigeration anesthesia was carried out in vivo fixation of Tenebrio molitor in-line phase contrast imaging, qualitative and quantitative analysis of the internal microstructure of insects in vivo. Through the improvement of experimental equipment, such as the use of fast imaging and sample detector before the shutter, and the combination of EST medium slope data acquisition mode Greatly reduced the number of projection, thereby reducing the radiation dose, the total exposure time acquisition of 320 projection experiments in the process of 1.92s, the radiation dose was 1.2 Gy, the dose is far less than the lethal dose of biological samples, after the experiment. The experimental data of Tenebrio molitor survived into the reconstruction phase and recovery phase recovery the three-dimensional structure information of 9 m pixel resolution. The 3D image reconstruction without phase recovery, because the effect caused by the Fresnel diffraction effect of edge enhancement can be observed within the trachea and tracheoles et al; and for Born algorithm by phase-attenuation-duality (PAD-BA) image reconstruction method of phase recovery, according to structure analysis of three dimensional refractive index. The spatial distribution of the quantitative experimental method used in this paper can not only get high resolution image reconstruction, at the same time Can greatly reduce the radiation dose of the sample, especially in biological imaging in vivo imaging have important applications. (3) of the imaging study of synchrotron radiation X ray phase contrast arthropod animal. Application of synchrotron radiation X ray phase contrast imaging technology of millimeter size of arthropod animals to carry out nondestructive three-dimensional structure research. In 12KeV energy collected from 450 angles of the head of the dry earthworm projection, single exposure time 6S, the projection without phase recovery and phase recovery projection algorithm using FBP 3D reconstruction were performed, qualitative and quantitative research on the earthworm external morphology and internal microstructure. According to the difference of specific structure and the density of different organizations, identify the internal microstructure of different, including muscle tissue, blood circulatory system, nervous system and digestive system. Compared with the commonly used method in tissue sections of the party Samples were stained, then by optical microscope or electron microscope observation, the kind of simple in-line phase contrast imaging method can get the sample of external morphology and internal microstructure of rapid nondestructive. Therefore, the experimental results proved the accuracy of the phase of X ray in the study of biological microstructure and high efficiency, and the potential applications in research of low density material. (4) research of micro CT image correction algorithm in shape artifacts. The ring artifact correction method can handle sinogram image while maintaining the authenticity of the effective removal of ring artifacts in CT images based on. This paper proposes and proves that a new calibration method ring artifact sinogram based processing. Unlike traditional method of sine graph processing, this method firstly in the sinogram positioning line like artifacts, and then the selected line in the original projection Artifact as noise through a filter to remove. Numerical simulation and drying of Tenebrio molitor synchrotron radiation X ray micro CT experimental data have shown that ring artifacts can be effectively removed and will not cause the loss of information structure, proved the feasibility and validity of this method. Compared with common sine graph method show that this method can get better the correction effect. This paper proposes a new perspective for ring artifact correction, by combining the linear artifact identification method and noise removal algorithm is more efficient and can get better ring artifact correction effect.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TH74;TH79
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本文编号：1432440