基于光纤束的共聚集荧光内窥成像研究
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摘要
由于人口增长和老龄化的加剧,以及抽烟,酗酒,缺乏锻炼,摄入高油脂食物等不良生活习惯,癌症近年来发生的比重越来越多。在发达国家癌症已经成为第一致死因素,在发展中国家癌症成为第二致死因素。研究表明,癌症多发生在上皮细胞的浅层,对癌症的诊断发现越早,成活率越高。所以对癌症的早期诊断已经越来越重要,对癌症早期诊断的各种设备也成为研究的热点。在这些成像诊断方法中,共聚焦荧光内窥系统可以在体实时提供亚细胞分辨率的结构信息,减少或者有可能取代组织活检,受到广泛的关注。本文就基于光纤束的共聚焦荧光内窥成像系统展开研究,并提出了基于傅里叶变换的多光谱荧光内窥成像系统。
本文研制开发了一种基于光纤束的共聚焦荧光内窥成像系统。并在光学结构,所用主要元件,控制程序和后期数据处理部分都进行了详细的讨论。在共聚焦荧光内窥成像系统中使用CRS共振型振镜实现扫描,取得4帧、秒1024×1024图像。在系统的控制电路中使用NI公司的商用采集卡和图形化编程软件Labview,使得我们能够关注系统的设计而不是繁琐的编程。首先使用标准分辨率板对其横向分辨率进行了测试得到其分辨率4.38微米。使用光栅样品测试系统成像区域为790微米。然后使用标准荧光小球和花粉颗粒检验了其对标准荧光样品的成像能力。最后对正常昆明小鼠的离体和在体组织进行了染色成像实验,验证了系统亚细胞分辨率对组织样品结构信息的分辨能力。
在多光谱荧光内窥成像系统中,我们结合了光纤束内窥成像的灵活性和傅里叶变换光谱仪的各种优势,可以在体对各种生物器官和组织进行高光谱分辨率的成像。在系统中我们设计使用了迈克尔逊干涉仪结构,选用精度较高的纳米位移台对运动平面镜进行移动。在系统中取出一部分照明光导入迈克尔逊干涉仪中实现对运动平面镜位置的实时监测。该种结构的设计可以保证系统的精确性和长期的稳定性。使用现有的平移台,理论上可以提供的最高光谱分辨率约是4.02cm-1。本文使用光谱稳定的氦氖激光器对其光谱分辨率进行了测量,和理论值有着较好的吻合。然后使用该系统对多色小球进行光谱成像验证了系统分辨多种荧光染料的能力。最后通过对深脑部位神经元进行光谱成像,展示了使用光纤束带来的内窥光谱成像能力。另外本文结合光纤束内窥功能和傅里叶变换光谱仪系统对标准样品和组织的激发光谱进行了测量,展示了使用激发光谱区分不同荧光基团的功能。
在共聚焦荧光内窥成像系统中使用光纤束可以带来成像的灵活性,但是光纤束本身也有蜂窝状的固有噪声。多种图像处理方法被提出用于消除光纤束的像素化。本文对视觉效果较好的自然邻点插值重建方法进行了论述。
With the aging and growth of the world population as well as an adoption of cancer causing behaviors, smoking, alcoholism,"westernized" diets, the incidence of cancer is increasing drasticly. Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. Studies found that the majority of cancers arise from malignant cells of epithelium. If diagnosed at early stage, patients will have a higher survival. So detection of cancer will be increasingly important, and multiple biomedical imaging techniques that can diagnose early stage cancer will be the focus of research. Confocal laser microendoscopy is a new tool that allow real time imaging at subcellular resolution during ongoing endoscopy, which reduce or eliminate the need for biopsy and histopathology. In this dissertation, the research focus on high resolution fluorescence microendoscopy and propose a multispectral imaging endo-spectroscopy that is based on fourier transform spectrometer.
A fiber bundle based high resolution fluorescence microendoscopy is constructed in this thesis. The optical lightpath, major component, control program, and data processing pipeline are discussed in this thesis. A CRS scanner is used in the high resolution fluorescence microendoscopy system to achieve a high imaging speed. A graphical programming language Labview and commercial commercial DAQ and image grab card are used, so the author can focus on the system design, not on the cockamamie programming. First we used the USAF target to verify the resolution of the system, then fluorescence beads and pollen were used to test the performance of the system. Excised tissue samples of normal Kunming mice were stained with0.1%acriflavine and imaged to demonstrate that the structure of tissue can be distinguished with subcellular resolution.
The multispectral imaging endo-spectroscopy system employs a flexible fiber bundle coupled to a home-built imaging Fourier transform spectroscope. The system retains several advantageous features of Fourier transform spectral measurement method, and can be used for in vivo imaging. A piezoelectric translation stage was used for moving the mirror, and a small portion of the excitation beam was reflected by a beam pickoff to monitor the moving mirror. The monitor channel guaranteed the accuracy of the FTS with long-term stability and eliminates extra spectral calibrations. Maximum spectral resolution of the system is4.02cm-1, very close to our measurement with a stabilized He-Ne laser. To validate its ability to distinguish multi-label objects, a mixture of10.0-14.0μm yellow and purple fluorescent microspheres was used in the experiment. At last, imaging experiment of dual-color cerebral cortex neural cells in vivo was performed to demonstrate the cerebral spectral imaging ability. In addition, we employ a fiber bundle and FTS to maesure the excitation spectrum of fluorescent microspheres and tissue to demonstrate the ability to to distinguish multi-label objects with excitation spectrum.
Endomicroscopy that emploies flexible fiber bundle can gain visual access to holes, hollows, and cavities that are difficult to enter and examine. However, fiber budle consists limited number of fibers, that leads to imaging artifacts, called comb structures. Many algorithms have been proposed for an effective removal of such artifacts. This thesis focus on the spatial interpolation method, which give a better visual effect.
本文研制开发了一种基于光纤束的共聚焦荧光内窥成像系统。并在光学结构,所用主要元件,控制程序和后期数据处理部分都进行了详细的讨论。在共聚焦荧光内窥成像系统中使用CRS共振型振镜实现扫描,取得4帧、秒1024×1024图像。在系统的控制电路中使用NI公司的商用采集卡和图形化编程软件Labview,使得我们能够关注系统的设计而不是繁琐的编程。首先使用标准分辨率板对其横向分辨率进行了测试得到其分辨率4.38微米。使用光栅样品测试系统成像区域为790微米。然后使用标准荧光小球和花粉颗粒检验了其对标准荧光样品的成像能力。最后对正常昆明小鼠的离体和在体组织进行了染色成像实验,验证了系统亚细胞分辨率对组织样品结构信息的分辨能力。
在多光谱荧光内窥成像系统中,我们结合了光纤束内窥成像的灵活性和傅里叶变换光谱仪的各种优势,可以在体对各种生物器官和组织进行高光谱分辨率的成像。在系统中我们设计使用了迈克尔逊干涉仪结构,选用精度较高的纳米位移台对运动平面镜进行移动。在系统中取出一部分照明光导入迈克尔逊干涉仪中实现对运动平面镜位置的实时监测。该种结构的设计可以保证系统的精确性和长期的稳定性。使用现有的平移台,理论上可以提供的最高光谱分辨率约是4.02cm-1。本文使用光谱稳定的氦氖激光器对其光谱分辨率进行了测量,和理论值有着较好的吻合。然后使用该系统对多色小球进行光谱成像验证了系统分辨多种荧光染料的能力。最后通过对深脑部位神经元进行光谱成像,展示了使用光纤束带来的内窥光谱成像能力。另外本文结合光纤束内窥功能和傅里叶变换光谱仪系统对标准样品和组织的激发光谱进行了测量,展示了使用激发光谱区分不同荧光基团的功能。
在共聚焦荧光内窥成像系统中使用光纤束可以带来成像的灵活性,但是光纤束本身也有蜂窝状的固有噪声。多种图像处理方法被提出用于消除光纤束的像素化。本文对视觉效果较好的自然邻点插值重建方法进行了论述。
With the aging and growth of the world population as well as an adoption of cancer causing behaviors, smoking, alcoholism,"westernized" diets, the incidence of cancer is increasing drasticly. Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. Studies found that the majority of cancers arise from malignant cells of epithelium. If diagnosed at early stage, patients will have a higher survival. So detection of cancer will be increasingly important, and multiple biomedical imaging techniques that can diagnose early stage cancer will be the focus of research. Confocal laser microendoscopy is a new tool that allow real time imaging at subcellular resolution during ongoing endoscopy, which reduce or eliminate the need for biopsy and histopathology. In this dissertation, the research focus on high resolution fluorescence microendoscopy and propose a multispectral imaging endo-spectroscopy that is based on fourier transform spectrometer.
A fiber bundle based high resolution fluorescence microendoscopy is constructed in this thesis. The optical lightpath, major component, control program, and data processing pipeline are discussed in this thesis. A CRS scanner is used in the high resolution fluorescence microendoscopy system to achieve a high imaging speed. A graphical programming language Labview and commercial commercial DAQ and image grab card are used, so the author can focus on the system design, not on the cockamamie programming. First we used the USAF target to verify the resolution of the system, then fluorescence beads and pollen were used to test the performance of the system. Excised tissue samples of normal Kunming mice were stained with0.1%acriflavine and imaged to demonstrate that the structure of tissue can be distinguished with subcellular resolution.
The multispectral imaging endo-spectroscopy system employs a flexible fiber bundle coupled to a home-built imaging Fourier transform spectroscope. The system retains several advantageous features of Fourier transform spectral measurement method, and can be used for in vivo imaging. A piezoelectric translation stage was used for moving the mirror, and a small portion of the excitation beam was reflected by a beam pickoff to monitor the moving mirror. The monitor channel guaranteed the accuracy of the FTS with long-term stability and eliminates extra spectral calibrations. Maximum spectral resolution of the system is4.02cm-1, very close to our measurement with a stabilized He-Ne laser. To validate its ability to distinguish multi-label objects, a mixture of10.0-14.0μm yellow and purple fluorescent microspheres was used in the experiment. At last, imaging experiment of dual-color cerebral cortex neural cells in vivo was performed to demonstrate the cerebral spectral imaging ability. In addition, we employ a fiber bundle and FTS to maesure the excitation spectrum of fluorescent microspheres and tissue to demonstrate the ability to to distinguish multi-label objects with excitation spectrum.
Endomicroscopy that emploies flexible fiber bundle can gain visual access to holes, hollows, and cavities that are difficult to enter and examine. However, fiber budle consists limited number of fibers, that leads to imaging artifacts, called comb structures. Many algorithms have been proposed for an effective removal of such artifacts. This thesis focus on the spatial interpolation method, which give a better visual effect.
引文
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