生物样品的数字全息显微相衬成像技术研究
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摘要
数字全息是传统光学全息术、计算机技术及光电探测技术相结合的产物，具有快速、实时、全视场、非接触和定量相衬成像的优势，尤其适合活体状态下的生物组织成像和测量。随着商品化的数字全息显微镜的面世，数字全息研究工作的重心开始由基础理论向发掘其潜在应用价值方面转移。目前无论是基础理论和算法的突破，还是应用领域的拓展，国内外对数字全息方法的研究工作都很活跃。
本文主要研究了数字全息显微相衬成像方法在生物组织成像方面的应用，在相衬成像关键算法、相干噪声抑制方法、数字全息显微系统光路结构和对生物组织成像中，得到了一系列理论和实验成果，主要完成了以下几个方面的工作：
1、研究了数字全息数值再现算法。在数字全息数值再现过程中，三种传统的数值传播算法分别是角谱算法、卷积算法和菲涅耳算法。用传统的数值传播算法，得到的再现像的像素大小、像素个数都是固定的，使其不适合应用于一些场合，如彩色数字全息、粒子场测量等，并有可能因此降低分辨率。针对该问题，我们提出了菲涅耳直接积分算法和瑞利-索末菲直接积分算法，使得再现平面的像素大小、像素数量和中心位置均可以调节，使数值再现过程更加灵活。应用所述的两种改进算法，我们实现了再现过程中人为设定再现像的像素大小、像素个数和中心位置，并在此基础上实现了分步再现后拼接。
2、研究了相位解包裹算法。在数字全息中，相位解包裹算法的优劣，直接决定了能否得到准确的相位图。我们结合一个统一的数学模型，对常用的相位解包裹算法进行了比较研究。随后结合离轴菲涅耳数字全息实验，提出了适合数字全息的相位解包裹算法，是基于最小二乘拟合的相位解包裹算法。
3、研究了相位畸变校正算法。在数字全息显微相衬成像过程中，总是存在相位畸变，而影响了正确物体相位信息的提取。现有的相位畸变校正方法，有的需要人工干预，有的需要精确测量系统参数。针对数字全息生物组织相衬显微成像，我们提出了一种基于改进数学模型的曲面拟合算法，可以自动去除主要相位畸变。该改进的数学模型，既包含一次项和二次项，又包含多个交叉项，可以更精确地对相位畸变规律进行拟合，并且可以用一个非迭代的算法进行计算。该计算方法大大简化了曲面拟合的计算过程，节省了再现过程中占用的计算资源。
4、研究了一种基于多步平均的倾斜照明相干噪声抑制方法。用多角度照明的方法，理论上能够通过多步平均抑制相干噪声，得到高质量再现像，然而其照明光路往往较复杂，且在相衬成像中对照明光斑大小和均匀度要求较高。我们提出了一种改进的多角度照明的相干噪声去除方法，克服了传统方法对照明光斑的大小和均匀度的要求。随后基于该方法搭建了一套结构紧凑的光纤无透镜傅里叶变换数字全息光路，能够自动获得多幅不同照明角度的数字全息图，经过平均再现算法后实现对相干噪声的抑制。
5、研究了一种透过高散射介质的成像方法。用可见光透过高散射或浑浊介质成像一直是一个重要的研究领域，在活体组织观测方面有潜在的应用价值。我们分析了光子穿过高散射介质的特性，提出一种基于短相干数字全息的成像方法，并利用多步散斑照明的方法抑制相干噪声。用一个低功率短相干激光二极管作为光源，搭建了光路，实验中实现了穿透3mm厚的鸡脯肉成像。
6、基于预放大离轴菲涅耳数字全息光路，搭建了一套小型化的光纤耦合倒置式数字全息显微镜系统。结合我们对数值再现算法、相位解包裹算法和相位畸变校正算法的研究，成功实现了对培养皿中贴壁生长的生物活细胞样品的相衬成像。
7、研究了数字全息显微成像中相位畸变的直接物理补偿方法。在数字全息显微相衬成像中，要进行频谱滤波、衍射传播、相位畸变校正和相位解包裹等一系列数据处理过程，影响了计算速度，需要尽可能较少计算负担，以利于长时间实时成像和观测。理论分析发现，通过合理的光路结构，可以在光路中直接补偿掉球面相位畸变，而不需要用专门的相位畸变校正算法。结合该理论分析，我们搭建了畸变实时去除数字全息显微系统，成功实现了对生物活细胞的长时间高精度观测。
Digital holography is the technology of the combination of the traditional opticalholography, computer technology and photoelectric detection technology withadvantages of fast, real-time, full field, non-contact and quantitative phase-contrastimaging. It is especially suitable for the imaging and measurement of biologicaltissues in vivo state. With the development of commercial digital holographicmicroscope, the research of digital holography began to move from basic theory toexploring its potential applications. At present, the research work in digitalholography is active at home and abroad both in basic theory and algorithm and theexpansion of application fields.
In this thesis, the topic is mainly about the application of digital holographicmicroscopy phase contrast imaging method in the imaging of biological samples. Inthe fields of key algorithms, coherent noise suppression methods, optical setups andimaging of biological samples in digital holographic microscopy, a series oftheoretical analysis and experimental results are given. They are depicted one by oneas follows:
1. The numerical reconstruction algorithms in digital holography are studied. Inthe numerical reconstruction of digital holography, there are three traditionalnumerical propagation algorithms: the angular spectrum algorithm, the convolutionalgorithm and the Fresnel algorithm. Using the traditional numerical propagationalgorithms, the pixel size, the number of pixels and the position of the reconstructedimage are fixed. So that it is not suitable for a number of occasions, such as colordigital holography, particle field measurements. To solve this problem, we proposeddirect integration Fresnel algorithm and direct integration Rayleigh-Sommerfeldalgorithm, with which the pixel size, the number of pixels, and the position of thereconstructed images are adjustable. It makes the numerical reconstruction moreflexible. Using the proposed algorithms, we adjusted the pixel size, the number ofpixels and the center position of the reconstructed experimental results. Based on this,we achieved a step-by-step reconstruction.
2. The phase unwrapping algorithms are studied. In digital holography, thequality of the reconstructed phase map depends on the phase unwrapping algorithm.With a unified mathematical model, a comparative study on the phase unwrappingalgorithms was done. On the basis of an off-axis Fresnel digital holographicexperiment, we concluded that the phase unwrapping algorithm based on least squaresfitting is appropriate in digital holography.
3. The phase aberration compensation algorithms are studied. In the phasecontrast imaging of digital holographic microscopy, phase aberrations always exist, which prevent to obtain the correct phase information. Some phase aberrationcompensation methods require manual intervention, while some others requireaccurate system parameters. In digital holographic biological phase-contrast imaging,we proposed a surface fitting algorithm based on an improved mathematical modelthat can remove the main phase aberrations automatically. The improvedmathematical model contains not only the first order and quadratic items, but alsosome cross ones, which can fit the phase aberration more accurately. It can beresolved by a non-iterative algorithm, which greatly simplifies the surface fittingprocess and eases the computational load.
4. A coherent noise suppression method based on the multiple obliqueilluminations is studied. The method based on the multiple oblique illuminations cansuppress the coherent noise and obtain high-quality image. However, the systems arealways complicated. We proposed an improved coherent noise suppression methodbased on the multiple oblique illuminations, which doesn’t demand a large anduniform light spot. Based on the proposed method we built up a compactfiber-coupled lensless Fourier transform digital holographic system. Multiple digitalholograms with different illumination angles can be obtained automatically, whichleads to suppression of the coherent noise by averaging.
5. A method for imaging through scattering media is studied. It is an importantarea of research to image through scattering or turbid media with visible light, and haspotential application values in biological imaging. Based on the analysis of thecharacteristic of photon passing though scattering media, we propose ashort-coherence digital holographic imaging method which utilize multi-step speckleillumination method to suppress the coherent noise. With a low-power shortcoherence laser diode, we achieved to image through3mm thick fresh chicken breast.
6. Based on the off-axis Fresnel digital holography with pre-magnification, acompact fiber-coupled inverted digital holographic microscopy system is built.Combined with our study on algorithms, we obtained phase-contrast imaging of livingcells adherent growing in the culture dish.
7. The physical compensation of phase aberrations in digital holographicmicroscopy is studied. In digital holography, it is necessary to ease computationalload as far as possible for long term real-time imaging and observation. Theoreticalanalysis shows that the spherical phase aberration could be compensated physicallywith proper optical setups. Subsequently, we built a phase aberration free digitalholographic microscope, and achieve long term observation of living cells with a highprecision.
本文主要研究了数字全息显微相衬成像方法在生物组织成像方面的应用，在相衬成像关键算法、相干噪声抑制方法、数字全息显微系统光路结构和对生物组织成像中，得到了一系列理论和实验成果，主要完成了以下几个方面的工作：
1、研究了数字全息数值再现算法。在数字全息数值再现过程中，三种传统的数值传播算法分别是角谱算法、卷积算法和菲涅耳算法。用传统的数值传播算法，得到的再现像的像素大小、像素个数都是固定的，使其不适合应用于一些场合，如彩色数字全息、粒子场测量等，并有可能因此降低分辨率。针对该问题，我们提出了菲涅耳直接积分算法和瑞利-索末菲直接积分算法，使得再现平面的像素大小、像素数量和中心位置均可以调节，使数值再现过程更加灵活。应用所述的两种改进算法，我们实现了再现过程中人为设定再现像的像素大小、像素个数和中心位置，并在此基础上实现了分步再现后拼接。
2、研究了相位解包裹算法。在数字全息中，相位解包裹算法的优劣，直接决定了能否得到准确的相位图。我们结合一个统一的数学模型，对常用的相位解包裹算法进行了比较研究。随后结合离轴菲涅耳数字全息实验，提出了适合数字全息的相位解包裹算法，是基于最小二乘拟合的相位解包裹算法。
3、研究了相位畸变校正算法。在数字全息显微相衬成像过程中，总是存在相位畸变，而影响了正确物体相位信息的提取。现有的相位畸变校正方法，有的需要人工干预，有的需要精确测量系统参数。针对数字全息生物组织相衬显微成像，我们提出了一种基于改进数学模型的曲面拟合算法，可以自动去除主要相位畸变。该改进的数学模型，既包含一次项和二次项，又包含多个交叉项，可以更精确地对相位畸变规律进行拟合，并且可以用一个非迭代的算法进行计算。该计算方法大大简化了曲面拟合的计算过程，节省了再现过程中占用的计算资源。
4、研究了一种基于多步平均的倾斜照明相干噪声抑制方法。用多角度照明的方法，理论上能够通过多步平均抑制相干噪声，得到高质量再现像，然而其照明光路往往较复杂，且在相衬成像中对照明光斑大小和均匀度要求较高。我们提出了一种改进的多角度照明的相干噪声去除方法，克服了传统方法对照明光斑的大小和均匀度的要求。随后基于该方法搭建了一套结构紧凑的光纤无透镜傅里叶变换数字全息光路，能够自动获得多幅不同照明角度的数字全息图，经过平均再现算法后实现对相干噪声的抑制。
5、研究了一种透过高散射介质的成像方法。用可见光透过高散射或浑浊介质成像一直是一个重要的研究领域，在活体组织观测方面有潜在的应用价值。我们分析了光子穿过高散射介质的特性，提出一种基于短相干数字全息的成像方法，并利用多步散斑照明的方法抑制相干噪声。用一个低功率短相干激光二极管作为光源，搭建了光路，实验中实现了穿透3mm厚的鸡脯肉成像。
6、基于预放大离轴菲涅耳数字全息光路，搭建了一套小型化的光纤耦合倒置式数字全息显微镜系统。结合我们对数值再现算法、相位解包裹算法和相位畸变校正算法的研究，成功实现了对培养皿中贴壁生长的生物活细胞样品的相衬成像。
7、研究了数字全息显微成像中相位畸变的直接物理补偿方法。在数字全息显微相衬成像中，要进行频谱滤波、衍射传播、相位畸变校正和相位解包裹等一系列数据处理过程，影响了计算速度，需要尽可能较少计算负担，以利于长时间实时成像和观测。理论分析发现，通过合理的光路结构，可以在光路中直接补偿掉球面相位畸变，而不需要用专门的相位畸变校正算法。结合该理论分析，我们搭建了畸变实时去除数字全息显微系统，成功实现了对生物活细胞的长时间高精度观测。
Digital holography is the technology of the combination of the traditional opticalholography, computer technology and photoelectric detection technology withadvantages of fast, real-time, full field, non-contact and quantitative phase-contrastimaging. It is especially suitable for the imaging and measurement of biologicaltissues in vivo state. With the development of commercial digital holographicmicroscope, the research of digital holography began to move from basic theory toexploring its potential applications. At present, the research work in digitalholography is active at home and abroad both in basic theory and algorithm and theexpansion of application fields.
In this thesis, the topic is mainly about the application of digital holographicmicroscopy phase contrast imaging method in the imaging of biological samples. Inthe fields of key algorithms, coherent noise suppression methods, optical setups andimaging of biological samples in digital holographic microscopy, a series oftheoretical analysis and experimental results are given. They are depicted one by oneas follows:
1. The numerical reconstruction algorithms in digital holography are studied. Inthe numerical reconstruction of digital holography, there are three traditionalnumerical propagation algorithms: the angular spectrum algorithm, the convolutionalgorithm and the Fresnel algorithm. Using the traditional numerical propagationalgorithms, the pixel size, the number of pixels and the position of the reconstructedimage are fixed. So that it is not suitable for a number of occasions, such as colordigital holography, particle field measurements. To solve this problem, we proposeddirect integration Fresnel algorithm and direct integration Rayleigh-Sommerfeldalgorithm, with which the pixel size, the number of pixels, and the position of thereconstructed images are adjustable. It makes the numerical reconstruction moreflexible. Using the proposed algorithms, we adjusted the pixel size, the number ofpixels and the center position of the reconstructed experimental results. Based on this,we achieved a step-by-step reconstruction.
2. The phase unwrapping algorithms are studied. In digital holography, thequality of the reconstructed phase map depends on the phase unwrapping algorithm.With a unified mathematical model, a comparative study on the phase unwrappingalgorithms was done. On the basis of an off-axis Fresnel digital holographicexperiment, we concluded that the phase unwrapping algorithm based on least squaresfitting is appropriate in digital holography.
3. The phase aberration compensation algorithms are studied. In the phasecontrast imaging of digital holographic microscopy, phase aberrations always exist, which prevent to obtain the correct phase information. Some phase aberrationcompensation methods require manual intervention, while some others requireaccurate system parameters. In digital holographic biological phase-contrast imaging,we proposed a surface fitting algorithm based on an improved mathematical modelthat can remove the main phase aberrations automatically. The improvedmathematical model contains not only the first order and quadratic items, but alsosome cross ones, which can fit the phase aberration more accurately. It can beresolved by a non-iterative algorithm, which greatly simplifies the surface fittingprocess and eases the computational load.
4. A coherent noise suppression method based on the multiple obliqueilluminations is studied. The method based on the multiple oblique illuminations cansuppress the coherent noise and obtain high-quality image. However, the systems arealways complicated. We proposed an improved coherent noise suppression methodbased on the multiple oblique illuminations, which doesn’t demand a large anduniform light spot. Based on the proposed method we built up a compactfiber-coupled lensless Fourier transform digital holographic system. Multiple digitalholograms with different illumination angles can be obtained automatically, whichleads to suppression of the coherent noise by averaging.
5. A method for imaging through scattering media is studied. It is an importantarea of research to image through scattering or turbid media with visible light, and haspotential application values in biological imaging. Based on the analysis of thecharacteristic of photon passing though scattering media, we propose ashort-coherence digital holographic imaging method which utilize multi-step speckleillumination method to suppress the coherent noise. With a low-power shortcoherence laser diode, we achieved to image through3mm thick fresh chicken breast.
6. Based on the off-axis Fresnel digital holography with pre-magnification, acompact fiber-coupled inverted digital holographic microscopy system is built.Combined with our study on algorithms, we obtained phase-contrast imaging of livingcells adherent growing in the culture dish.
7. The physical compensation of phase aberrations in digital holographicmicroscopy is studied. In digital holography, it is necessary to ease computationalload as far as possible for long term real-time imaging and observation. Theoreticalanalysis shows that the spherical phase aberration could be compensated physicallywith proper optical setups. Subsequently, we built a phase aberration free digitalholographic microscope, and achieve long term observation of living cells with a highprecision.
引文
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