光学显微成像及在生物样品显示与测量中的应用
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摘要
生命科学研究的突破在很大程度上依赖于各种先进的分析测试系统,显微成像系统是其中最常见和最重要的分析测试系统之一。传统的显微镜已经远不能满足目前生命科学的要求,发展新的显微成像分析技术,对细胞等微小生物样品实现无创伤、定量、动态的显微分析是目前生命科学研究的迫切需求。本文分别基于干涉和无透镜全息原理,发展了两种新型光学显微技术,来实现对生物样品的成像与测量,取得的主要研究成果有：
在广泛调研生物显微技术的基础上,设计并研制了一套离轴干涉显微成像系统,以血红细胞为样品,实现了其光相位场的定量成像。干涉图的处理选择以傅里叶变换为基础的相位提取技术和基于离散余弦变换的无加权最小二乘法的相位解包技术,得到的光相位场分布结果与文献报道吻合。
利用上述研制的干涉显微系统,首次通过相位成像分析了金属离子对细胞的影响,给出了细胞受影响后的定量相位分布,计算出相位面积和相位体积等参数,实现了对血红细胞状态的实时、定量的全程分析,并由此判定金属离子对细胞的破坏趋势和程度。所得的定量分析结果与微分干涉差(DIC)显微镜的定性观测一致。
为了得到被测生物样品更多的细节信息,进一步研究了微离轴干涉模式,提出了将希尔伯特变换相位提取算法引入到微离轴单幅干涉图的处理中,有效改善了波前恢复的高频分量,使细胞轮廓更加清晰。通过直径、面积、圆度和相位分布直方图等参数评价基于微离轴干涉模式的希尔伯特定量相位成像系统的性能,结果表明该系统在灵敏度和边缘轮廓提取等方面具有较强的优势,兼备快速采样和多细节成像能力。
为了突破现有显微成像技术视场的瓶颈,研究了无透镜全息显微成像术,其视场大小和记录用数字传感器的有效感光面积一样。为保证大视场前提下的高分辨率,引入了亚像元高分辨率成像术,实现了单幅图视场达18cm2的1.5G(等效)像素成像,半节距分辨率达2.19μmn。
大视场成像为跟踪动态生物样品的运动轨迹提供了便利,据此发展了双光源无透镜全息显微成像系统,同时追踪了成千上万的精子的三维运动轨迹,揭示了部分精子游动服从螺旋型运动规律,并且右手型螺旋运动数目占总螺旋型数目的90%以上,给出了精子运动线速度、角速度等运动参数。在此基础上,研制了便携式双光源无透镜全息显微成像系统,具有体积小,质量轻等特点,在远程医疗中具有潜在应用价值。
To a great degree, the breakthrough in the field of life science depends on various novel and powerful measurement systems. The microscopy apparatus is the most widely used and important device for magnifying imaging. However, the conventional microscope cannot meet the rapid development of the scientific research. Therefore, it is necessary to grope for innovative microscopic imaging tools that can realize real-time, non-invasive and quantitative analysis. In this dissertation, two kinds of optical microscopy imaging techniques are developed based on interferometry and lensfree holography, to realize bio-sample display and measurement. The detailed contents can be generalized as:
A interferometric phase microscopy imaging system is proposed based on extensive research literature on bio-microscopy techniques. Red blood cells (RBCs) are samples and used for phase imaging. The captured interferograms are processed via fast Fourier transform based phase retrieval method and2D discrete cosine transform unweighted least-squares algorithm based phase unwrapping method. The phase distributions of RBCs are recovered quantitatively, which agree with the literature findings.
Infection of cells by metallic ions leads to both biochemical and structural modifications, and interferometric phase microscopy is adopted for the first time to detect such effects. The phase distributions of RBCs damaged by the lithium and lead ions are recovered in a real-time, quantitative and whole-process analytical manner, which are identical with the cell shape and qualitative phase distribution observation under differential interference contrast (DIC) microscopy. Furthermore, accurate phase area and phase volume values could be calculated. The results could be used for damage estimation and trend assessment of infected cells.
A slightly-off-axis interferometry is further discussed to acquire more details on biosamples. Hilbert phase microscopy (HPM) method is developed as a phase retrieval approach in the post-processing with more information about the cells edge and high frequency components. Slightly-off-axis interferometry based HPM is evaluated by parameters such as diameter, phase area, phase volume and phase distribution histogram. The experimental results show that the proposed method owns fine spatial details and real-time imaging capability, which are useful for cells detection.
Field of view (FOV) of system is one of the key parameters in the microscopic imaging. In this system, FOV is the same size as the effective photosensitive imaging area of the image sensor. For each lensfree hologram, the pixel size of sensor chip limits the spatial resolution of the reconstructed image. To circumvent this limitation, a sub-pixel shifting super-resolution algorithm is implemented to improve the resolution. Gigapixel imaging is achieved based on such a platform with a FOV of～18cm2and2.19μm half-pitch resolution.
Wide FOV provides a platform for tracking bio-samples trajectories. Dual-view lensfree on-chip imaging technique that can track3D trajectories of thousands of individual human sperms is demonstrated. The large statistics provided by this lensfree imaging platform reveal that rare motile human sperms swim along well-defined helices. Furthermore, among these observed helical human sperms, approximately90%prefer right-handed helices over left-handed ones. What's more, some parameters such as helical rotation speed and linear speed can be determined. The portable lab-on-a-chip device is manufactured with unique features like compactness and light weight. It could in general be quite valuable for telemedicine.
在广泛调研生物显微技术的基础上,设计并研制了一套离轴干涉显微成像系统,以血红细胞为样品,实现了其光相位场的定量成像。干涉图的处理选择以傅里叶变换为基础的相位提取技术和基于离散余弦变换的无加权最小二乘法的相位解包技术,得到的光相位场分布结果与文献报道吻合。
利用上述研制的干涉显微系统,首次通过相位成像分析了金属离子对细胞的影响,给出了细胞受影响后的定量相位分布,计算出相位面积和相位体积等参数,实现了对血红细胞状态的实时、定量的全程分析,并由此判定金属离子对细胞的破坏趋势和程度。所得的定量分析结果与微分干涉差(DIC)显微镜的定性观测一致。
为了得到被测生物样品更多的细节信息,进一步研究了微离轴干涉模式,提出了将希尔伯特变换相位提取算法引入到微离轴单幅干涉图的处理中,有效改善了波前恢复的高频分量,使细胞轮廓更加清晰。通过直径、面积、圆度和相位分布直方图等参数评价基于微离轴干涉模式的希尔伯特定量相位成像系统的性能,结果表明该系统在灵敏度和边缘轮廓提取等方面具有较强的优势,兼备快速采样和多细节成像能力。
为了突破现有显微成像技术视场的瓶颈,研究了无透镜全息显微成像术,其视场大小和记录用数字传感器的有效感光面积一样。为保证大视场前提下的高分辨率,引入了亚像元高分辨率成像术,实现了单幅图视场达18cm2的1.5G(等效)像素成像,半节距分辨率达2.19μmn。
大视场成像为跟踪动态生物样品的运动轨迹提供了便利,据此发展了双光源无透镜全息显微成像系统,同时追踪了成千上万的精子的三维运动轨迹,揭示了部分精子游动服从螺旋型运动规律,并且右手型螺旋运动数目占总螺旋型数目的90%以上,给出了精子运动线速度、角速度等运动参数。在此基础上,研制了便携式双光源无透镜全息显微成像系统,具有体积小,质量轻等特点,在远程医疗中具有潜在应用价值。
To a great degree, the breakthrough in the field of life science depends on various novel and powerful measurement systems. The microscopy apparatus is the most widely used and important device for magnifying imaging. However, the conventional microscope cannot meet the rapid development of the scientific research. Therefore, it is necessary to grope for innovative microscopic imaging tools that can realize real-time, non-invasive and quantitative analysis. In this dissertation, two kinds of optical microscopy imaging techniques are developed based on interferometry and lensfree holography, to realize bio-sample display and measurement. The detailed contents can be generalized as:
A interferometric phase microscopy imaging system is proposed based on extensive research literature on bio-microscopy techniques. Red blood cells (RBCs) are samples and used for phase imaging. The captured interferograms are processed via fast Fourier transform based phase retrieval method and2D discrete cosine transform unweighted least-squares algorithm based phase unwrapping method. The phase distributions of RBCs are recovered quantitatively, which agree with the literature findings.
Infection of cells by metallic ions leads to both biochemical and structural modifications, and interferometric phase microscopy is adopted for the first time to detect such effects. The phase distributions of RBCs damaged by the lithium and lead ions are recovered in a real-time, quantitative and whole-process analytical manner, which are identical with the cell shape and qualitative phase distribution observation under differential interference contrast (DIC) microscopy. Furthermore, accurate phase area and phase volume values could be calculated. The results could be used for damage estimation and trend assessment of infected cells.
A slightly-off-axis interferometry is further discussed to acquire more details on biosamples. Hilbert phase microscopy (HPM) method is developed as a phase retrieval approach in the post-processing with more information about the cells edge and high frequency components. Slightly-off-axis interferometry based HPM is evaluated by parameters such as diameter, phase area, phase volume and phase distribution histogram. The experimental results show that the proposed method owns fine spatial details and real-time imaging capability, which are useful for cells detection.
Field of view (FOV) of system is one of the key parameters in the microscopic imaging. In this system, FOV is the same size as the effective photosensitive imaging area of the image sensor. For each lensfree hologram, the pixel size of sensor chip limits the spatial resolution of the reconstructed image. To circumvent this limitation, a sub-pixel shifting super-resolution algorithm is implemented to improve the resolution. Gigapixel imaging is achieved based on such a platform with a FOV of～18cm2and2.19μm half-pitch resolution.
Wide FOV provides a platform for tracking bio-samples trajectories. Dual-view lensfree on-chip imaging technique that can track3D trajectories of thousands of individual human sperms is demonstrated. The large statistics provided by this lensfree imaging platform reveal that rare motile human sperms swim along well-defined helices. Furthermore, among these observed helical human sperms, approximately90%prefer right-handed helices over left-handed ones. What's more, some parameters such as helical rotation speed and linear speed can be determined. The portable lab-on-a-chip device is manufactured with unique features like compactness and light weight. It could in general be quite valuable for telemedicine.
引文
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