非线性光学显微镜的研究

【作者】 郭利娜；

【导师】 唐志列；

【作者基本信息】 华南师范大学 ， 光学， 2007， 硕士

【摘要】 非线性共焦显微镜，如双光子荧光(TPF)和多光子荧光(MPF)共焦显微镜、二次谐波(SHG)和三次谐波(THG)共焦显微镜、相干反斯托克斯散射(CARS)共焦显微镜等，是基于非线性光学效应的高分辨率成像技术。非线性效应能导致其成像焦斑比激发光的焦斑更小，从而突破了经典衍射极限，极大地改善了共焦显微镜的空间分辨率，从而实现超分辨率三维成像。非线性显微镜具有空间分辨率高、层析成像、穿透深度大等优点，因而在生物医学、生命科学、材料科学等领域具有变革性的应用潜力。近二十年来，非线性共焦显微镜的理论和实验研究取得了许多突破性进展，已成为光学及其交叉学科中最诱人、最活跃的研究领域之一。而拉曼感生克尔效应光谱(RIKES)是一种三阶非线性相干拉曼光谱学过程，大多数文献只是对如何提高RIKES检测的灵敏度做了理论和实验研究，但能否用RIKES光谱来进行显微成像，目前还没有文献做过系统的理论分析。RIKES成像不同于单光束成像模式，它需要采用两束光进行成像：即泵浦光和探测光，强的泵浦光会对弱的探测光(成像光束)产生很强的非线性调制作用，当泵浦光与探测光的频率差刚好等于样品的某个拉曼模的频率时，强的泵浦光不仅会引起成像光束的锐化，还会引起成像光束偏振态的改变。因此，RIKES成像除了具有非线性共焦显微镜的上述优点外，包含了更多物质内部的结构信息，如：分子的振动模、振动取向以及光克尔效应等信息，其次，RIKES成像是特征成像，那么，对分子某些特征的振动模式进行成像，能够实现分子的特征识别，因此，在分子影像和单分子检测方面具有重要意义。本文提出了基于拉曼感生克尔效应光谱(RIKES)的非线性共焦显微镜的和微分共焦显微镜的成像模式，从理论上论证了RIKES成像的可行性，研究了RIKES非线性共焦显微镜的三维成像特性，结果表明：RIKES成像具有内在的光学层析成像能力和特征成像能力，可以突破经典衍射极限，实现超分辨率特征成像，有望发展成为一种新型的可与目前其它的非线性成像模式相媲美的非线性成像技术。本文的主要内容包括：第一、研究了RIKES的物理机制，提出利用RIKES光谱进行显微成像的新型的成像模式，在理论上验证了该成像模式的可行性，系统地研究了拉曼感生克尔效应光谱成像的特点。第二、利用傅立叶光学成像理论和非线性原理，研究了RIKES共焦显微镜的成像理论。在此基础上，分析了RIKES非线性共焦显微镜的三维成像特性，最后推导了RIKES共焦显微镜的光学传递函数。第三、推导了高斯光束非线性拉曼共焦显微镜的成像理论。同时，与点光源描述的非线性拉曼共焦显微镜的点扩散函数相比，两者具有内在的一致性。在此基础上，研究了泵浦光对探测光的空间调制机制，分析了不同束腰尺寸的泵浦光和探测光对非线性拉曼共焦显微镜空间分辨率的影响。第四、提出了一种利用时间分辨技术结合共焦扫描实现微分共焦显微镜的新的方法，推导了微分共焦显微镜的成像理论。结果表明，微分共焦显微镜，可以突破经典衍射极限，极大地改善共焦显微镜的横向分辨率，从而实现超分辨率微分成像。更多还原

【Abstract】 Nonlinear confocal microscopy, such as two-photon fluorescence （TPF） and multi-photon fluorescence （MPF）, second-harmonic generation （SHG） and third-harmonic generation （THG）, Coherent anti-Stoke Raman Scattering （CARS） microscopy, has been demonstrated the potential applications in biomedicine, biology and material science et al. As a powerful optical imaging technique, nonlinear confocal microscopy intrinsically provides three-dimensional （3-D） imaging capability and potentially produces image contrast with chemical and physical specificity inside scattering samples. However, is it feasible to utilize Raman induced Kerr effect spectroscopy （RIKES） imaging? No person studies it systematically.In RIKES imaging, a strong circularly polarized pump beam at frequencyω₁ and a weak, y-polarized bean at frequencyω_s are employed. The pump beam modulated nonlinearly on the probe beam, i.e., the imaging beam. When the frequency differenceω_l-ω_s is tuned to match the frequency of a Raman modeω_R, i.e.,ω_l-ω_s=ω_R, the pump beam can induce an enhanced circular birefringence, which consequently causes a polarization rotation of the linearly polarized probe beam, termed Raman induced Kerr effect spectroscopy （RIKES）. Beside the above advantages of nonlinear confocal microscopy, RIKES imaging is capable to characterize inherent structural features, such as vibration mode, vibration orientation, and optically-induced molecular reorientation et al. Considering that Raman spectrum is the fingerprint of the molecular structure, So RIKES imaging on the vibration mode can provide the basis for molecular identification, which is significant in molecular imaging and single molecular detection.If RIKES imaging is utilized instead of two-photon fluorescence, SHG and CARS imaging, it has potential to provide a novel sub-diffraction limit characteristic imaging method comparable to the existing imaging techniques based on other nonlinear optical processes, such as two-photon fluorescence, SHG and CARS.In this paper, a novel nonlinear confocal microscopy that utilizing Raman induced Kerr effect spectroscopy （RIKES） is presented. The imaging theory of RIKES confocal microscopy is derived. The imaging properties of RIKES confocal microscopy has been analyzed in detail. The main contents are as follows:Firstly, the physical mechanism of RIKES generation is discussed. A novel nonlinear imaging utilizing RIKES is presented. The characteristic of RIKES imaging is analyzed in detail.Secondly, according to Fourier imaging theory and nonlinear optical principle, the imaging theory of RIKES nonlinear confocal microscopy is derived. With the imaging theory, the impact of nonlinear property of RIKES on the spatial resolution and imaging properties of confocal microscopy has been analyzed in detail. Aditionally, the three-dimensional optical transfer function （OTF） of RIKES nonlinear confocal microscopy is calculated.Thirdly, considering the Gaussian distribution of source function, the three-dimensional point spread function of RIKES confocal microscopy is derived. The spatial modulation mechanism of pump beam on the probe beam is discussed.Finally, a novel differential confocal microscopy is developed based on a time-resolved technique and confocal scanning technique. The imaging theory of differential confocal microscopy is derived. The three-dimensional point spread function of differential confocal microscopy is derived. It is shown the differential confocal imaging technique can break through the classic diffraction limit and achieve high-resolution imaging.更多还原