本文關(guān)鍵詞：基于同步輻射X射線的多尺度生物成像研究　出處：《山東大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 生物成像 低輻射劑量 多尺度 同步輻射X射線 斷層掃描成像

【摘要】：對(duì)生物顯微結(jié)構(gòu)的認(rèn)識(shí)橫跨多個(gè)尺度,包括生物個(gè)體、組織到細(xì)胞等。實(shí)現(xiàn)多尺度的生物系統(tǒng)的成像對(duì)于生物科學(xué)的發(fā)展有重要的意義。x射線因其波長(zhǎng)短和穿透能力強(qiáng)可以實(shí)現(xiàn)多尺度生物樣品的無損、三維、高分辨成像,彌補(bǔ)了光學(xué)顯微鏡和電子顯微鏡在生物成像中存在的不足。隨著第三代同步輻射X射線光源和光學(xué)器件制作工藝的發(fā)展,X射線成像技術(shù)逐漸成為生物結(jié)構(gòu)研究的重要工具。但是,在X射線成像方法及應(yīng)用中仍然存在一定的問題,包括生物樣品在成像過程中的輻射損傷問題,三維重建算法的精確程度,以及三維重建圖像的后期圖像處理等。針對(duì)X射線成像中存在的科學(xué)問題,本文開展了基于同步輻射X射線的多尺度生物成像方法學(xué)探索,并在此基礎(chǔ)上對(duì)細(xì)胞及昆蟲等生物樣品的三維高分辨成像進(jìn)行了應(yīng)用研究。具體內(nèi)容包括：(1)基于同步輻射X射線的三維、高分辨、低劑量細(xì)胞成像和細(xì)胞內(nèi)納米材料的雙能襯度X射線成像方法及應(yīng)用研究。結(jié)合掃描透射X射線成像技術(shù)(Scanning Transmission X-ray Microscopy, STXM)和等斜率層析算法(Equally Sloped Tomography, EST)實(shí)現(xiàn)對(duì)[Gd@C82(OH)22]n溶液中培養(yǎng)的貼壁巨噬細(xì)胞的三維高分辨成像。在Gd的M2吸收邊前(能量1186eV)和吸收邊上(能量1189eV)按等斜率方式分別采集了兩套數(shù)據(jù)(每套包含46個(gè)投影,角度分布±79.4°)。通過EST重建獲得巨噬細(xì)胞在50 nm像素分辨率下的三維顯微結(jié)構(gòu)。觀察到細(xì)胞的特征性表面形貌,例如扁平的三維結(jié)構(gòu),伸出的觸手；識(shí)別出細(xì)胞核、溶酶體等不同的細(xì)胞器。通過在實(shí)驗(yàn)過程中采用掃描成像模式和三維重建過程中采用新的圖像重建算法,使得生物樣品的輻射劑量大幅度降低到4.43×106 Gy。根據(jù)已有研究,該輻射劑量不會(huì)引起細(xì)胞結(jié)構(gòu)的改變；同時(shí),通過對(duì)比實(shí)驗(yàn)前后細(xì)胞在0°的投影,進(jìn)一步證實(shí)了結(jié)構(gòu)沒有發(fā)生變化。等斜率迭代算法的應(yīng)用有效地解決了實(shí)驗(yàn)數(shù)據(jù)有限,以及采集過程中高角度投影缺失的問題,同時(shí)提高了三維圖像重建的準(zhǔn)確度,有利于得到細(xì)胞的精確三維結(jié)構(gòu)。系統(tǒng)研究了雙能襯度x射線成像技術(shù),根據(jù)x射線的吸收在特定元素吸收邊能量前后發(fā)生突變的特性,實(shí)現(xiàn)了巨噬細(xì)胞內(nèi)納米材料[Gd@C82(OH)22]n的三維定量成像。分析對(duì)比了雙能襯度成像方法中兩種計(jì)算三維元素分布的途徑：第一種,首先計(jì)算所有角度的二維元素分布,然后經(jīng)旋轉(zhuǎn)軸校正和強(qiáng)度歸一化后進(jìn)行三維重建,得到三維元素分布；第二種,先分別對(duì)兩個(gè)能量的數(shù)據(jù)進(jìn)行旋轉(zhuǎn)軸校準(zhǔn)和強(qiáng)度的校正,然后進(jìn)行三維重建,得到兩個(gè)能量對(duì)應(yīng)的三維圖像,隨后配準(zhǔn)兩個(gè)三維圖像,計(jì)算二者差異得到三維元素分布。對(duì)比發(fā)現(xiàn)第二種方法可以得到更好的圖像效果,有更高的信噪比,同時(shí)第二種更簡(jiǎn)單易于操作�；诘诙N方法得到的結(jié)果,分析了巨噬細(xì)胞內(nèi)Gd元素(即[Gd@C82(OH)22]n)的三維空間分布,與同一細(xì)胞的x射線熒光成像(X-Ray Fluorescence, XRF)相比具有更好的圖像質(zhì)量和分辨能力。實(shí)驗(yàn)結(jié)果顯示[Gd@C82(OH)22]n發(fā)生聚集并被巨噬細(xì)胞大量吞入,主要分布在溶酶體內(nèi),但并未進(jìn)入細(xì)胞核。其它相同條件培養(yǎng)得到的細(xì)胞也證實(shí)了納米材料在巨噬細(xì)胞內(nèi)具有類似的空間分布。對(duì)巨噬細(xì)胞的形貌、細(xì)胞內(nèi)納米材料的空間分布以及生理分析將有助于理解[Gd@C82(OH)22]n這種潛在的納米藥物在體內(nèi)和體外均顯示高效低毒的抗腫瘤效果。同時(shí),結(jié)合EST算法和STXM對(duì)貼壁生長(zhǎng)的Ag標(biāo)定的淋巴細(xì)胞進(jìn)行了三維成像研究。在1060 eV采集了37個(gè)等斜率投影,角度分布為-63.44°到69.44°,聚焦光斑直徑50 nm,掃描步進(jìn)50 nm。通過EST重建得到細(xì)胞的三維表面形貌,尺寸-20x17×5μm3,但內(nèi)部結(jié)構(gòu)信息不易分辨。主要原因是細(xì)胞的厚度超出了波帶片的焦深范圍,造成圖像的模糊,無法識(shí)別細(xì)胞內(nèi)的精細(xì)結(jié)構(gòu)；1060 eV的軟x射線對(duì)于樣品的透過率很低,造成高密度或者較厚的區(qū)域無法分辨。另外,由于標(biāo)定的Ag顆粒尺寸遠(yuǎn)小于成像分辨率,目前無法根據(jù)吸收襯度的差異實(shí)現(xiàn)對(duì)單個(gè)顆粒成像識(shí)別。通過對(duì)成像參數(shù)例如能量、樣品厚度、波帶片焦深等對(duì)三維成像質(zhì)量影響的分析,增進(jìn)了對(duì)于細(xì)胞三維成像方法的理解,對(duì)生物樣品成像實(shí)驗(yàn)提供了指導(dǎo)。(2)活體昆蟲的同步輻射X射線相位襯度成像研究。研究包括兩個(gè)方面,首先通過對(duì)干燥黃粉蟲吸收襯度成像,驗(yàn)證EST算法的高效和準(zhǔn)確性；然后對(duì)活體黃粉蟲進(jìn)行同軸相位襯度X射線成像。首先,對(duì)干燥的黃粉蟲進(jìn)行同步輻射X射線吸收襯度顯微成像,在12 KeV采集了320個(gè)等斜率投影和600個(gè)等角度投影,單張曝光55 ms,分別用EST算法和濾波反投影算法(Filtered Back Projection algorithm, FBP)進(jìn)行圖像重建。通過改變兩種算法在重建過程中所用的投影數(shù)目,系統(tǒng)地對(duì)比了兩種重建算法的三維重建效果。信噪比、襯度和噪聲功率譜等圖像參數(shù)都顯示采用EST算法時(shí)320個(gè)投影得到的圖像重建效果遠(yuǎn)遠(yuǎn)好于采用FBP算法時(shí)600個(gè)投影的重建效果。即使用180個(gè)投影,采用EST算法也可以得到顯微結(jié)構(gòu)可識(shí)別的三維圖像。對(duì)比結(jié)果顯示EST算法可以在保持分辨率的情況下減少三分之二的投影數(shù)目。投影數(shù)目的減少可以有效降低生物樣品的輻射劑量。根據(jù)EST算法的重建圖像分析了干燥黃粉蟲的三維表面形貌和內(nèi)部顯微結(jié)構(gòu),如肌肉組織等。利用同步輻射X射線,對(duì)冷凍麻醉固定活體黃粉蟲開展了同軸相位襯度成像,定性和定量地分析昆蟲在活體狀態(tài)的內(nèi)部顯微結(jié)構(gòu)。通過實(shí)驗(yàn)設(shè)備的改進(jìn),如快速成像探測(cè)器的使用和樣品前快門的使用,并結(jié)合EST中等斜率數(shù)據(jù)采集模式極大地減少了投影數(shù)目,從而降低了輻射劑量,實(shí)驗(yàn)過程中采集320個(gè)投影的總曝光時(shí)間為1.92s,輻射劑量為1.2 Gy,該劑量遠(yuǎn)遠(yuǎn)小于生物樣品的致死劑量,實(shí)驗(yàn)后黃粉蟲仍然存活。對(duì)實(shí)驗(yàn)數(shù)據(jù)分別進(jìn)行了相位恢復(fù)前和相位恢復(fù)后的三維重建,得到9μm像素分辨率的三維結(jié)構(gòu)信息。對(duì)未做相位恢復(fù)的三維圖像重建,由于菲涅耳衍射效應(yīng)造成的邊緣增強(qiáng)效果,可以觀測(cè)到內(nèi)部氣管及微氣管等組織；而對(duì)于采用phase-attenuation-duality Born algorithm (PAD-BA)方法相位恢復(fù)后重建的圖像,可根據(jù)三維折射率的空間分布進(jìn)行定量的結(jié)構(gòu)分析。本論文中采用的實(shí)驗(yàn)方法不僅可得到高分辨三維重建圖像,同時(shí)還可以極大地減少樣品的輻射劑量,在生物成像尤其是活體成像中會(huì)有重要的應(yīng)用。(3)節(jié)肢動(dòng)物的同步輻射X射線相位襯度成像研究。應(yīng)用同步輻射X射線同軸相位襯度成像技術(shù)對(duì)毫米尺寸的節(jié)肢動(dòng)物開展了無損三維結(jié)構(gòu)研究。在12KeV能量下采集了干燥蚯蚓頭部的450個(gè)等角度投影,單張曝光時(shí)間6s,利用FBP算法對(duì)未做相位恢復(fù)的投影和已做相位恢復(fù)的投影,分別進(jìn)行三維重建,定性和定量地研究了蚯蚓的外部形貌和內(nèi)部顯微結(jié)構(gòu)。根據(jù)不同組織的特異性結(jié)構(gòu)和密度的差異,識(shí)別出來不同的內(nèi)部顯微組織,包括肌肉組織、血液循環(huán)系統(tǒng)、神經(jīng)系統(tǒng)和消化系統(tǒng)。相對(duì)于常用的組織切片方法中對(duì)樣品進(jìn)行切片染色處理,然后用光學(xué)顯微鏡或電子顯微鏡觀測(cè),該種簡(jiǎn)易的同軸相位襯度成像方法可快速無損地得到樣品的外部形貌和內(nèi)部顯微結(jié)構(gòu)。因此,實(shí)驗(yàn)結(jié)果充分證明了相位襯度X射線在生物顯微結(jié)構(gòu)研究中的準(zhǔn)確性和高效性,以及在研究低密度材料中的應(yīng)用潛力。(4)顯微CT圖像中環(huán)形偽影的校正算法研究。基于正弦圖處理的環(huán)形偽影校正方法可以在保持圖像真實(shí)性的同時(shí)有效去除CT圖像中的環(huán)形偽影。本論文提出并證實(shí)了一種基于正弦圖處理的環(huán)形偽影校正新方法。和傳統(tǒng)的正弦圖處理的方法不同,本方法首先在正弦圖中定位出線狀偽影,然后在原始投影中把選出的線狀偽影作為點(diǎn)噪音通過濾波去除。數(shù)值模擬和干燥黃粉蟲的同步輻射X射線顯微CT實(shí)驗(yàn)數(shù)據(jù)都顯示環(huán)形偽影可以有效的去除而不會(huì)引起結(jié)構(gòu)信息的丟失,證實(shí)了該方法的可行性和有效性。和常用正弦圖方法的對(duì)比顯示本文方法可以得到更好的校正效果。本文提出了一個(gè)環(huán)形偽影校正的新視角,通過結(jié)合更高效的線狀偽影識(shí)別方法和點(diǎn)噪音去除算法,可以得到更好的環(huán)形偽影校正效果。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：TH74;TH79
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本文編號(hào)：1432440