高分辨受激拉曼光谱显微成像及应用研究

发布时间：2018-03-11 06:05

本文选题：非线性光学显微成像　切入点：无标记成像　出处：《哈尔滨工业大学》2016年博士论文　论文类型：学位论文

【摘要】：受激拉曼散射(stimulated Raman scattering,SRS)显微成像技术是一种新兴的无标记显微成像技术,其成像衬度来源于分子振动特性,因而具有优异的化学选择性和化学特异性。除此之外,由于SRS显微成像具有高分辨率、高灵敏度和无侵入性等特点,在不到十年的时间里已经发展成为生命科学领域研究中的一项重要工具。基于高光谱SRS和多元SRS的受激拉曼光谱显微成像更是在每一个像素点都拥有一段光谱,因此其不仅可以区分拥有重叠拉曼谱带的不同分子,还能提供更加丰富的化学信息,成为最近两三年来研究的前沿和热点。然而受发展时间限制,目前受激拉曼光谱显微成像的研究还有很多方面需要完善,其应用仍然需要开拓。鉴于此,本文利用光谱聚焦以及非线性光谱压缩和脉冲整形相结合的方法开展了高分辨受激拉曼光谱成像技术以及其在膜电位检测和木质素化学成分分析中的应用研究。理论上,本文首先利用非线性耦合波方程对SRS信号的产生过程进行了推导,给出了SRS信号的表达式。分析并比较了不同非共振背景下的SRS和CARS光谱线型的差异。研究了入射激光偏振对不同退偏比拉曼振动模SRS信号强度的影响,揭示了偏振调制SRS成像的基本原理。详细讨论了SRS成像中的噪声、信噪比以及背景等问题,指出在理想情况下SRS显微成像系统应具有散粒噪声极限探测能力。分析了光谱聚焦方法实现高光谱相干拉曼散射显微成像的机理,并利用玻璃棒引入线性啁啾建立了一套基于光谱聚焦方法的高光谱SRS显微成像系统,针对该方法中存在的拉曼频移校准和信号强度校准问题,给出了具体的解决方案。同时在应用研究方面,首次探讨了利用SRS信号无标记探测细胞膜电位的可行性。以红细胞血影为模型,完成了单个自然细胞膜的振动光谱成像,证实了高光谱SRS显微成像探测单个细胞膜的灵敏度。通过操控细胞膜内外离子成分改变跨膜电位,同时利用受激拉曼光谱成像观察不同电势下的细胞膜,发现SRS光谱线型随膜电位改变发生显著变化,结果表明SRS成像有望用于细胞膜电位的无标记测量。考虑光谱聚焦方法光谱分辨率较差,难以满足指纹区的成像应用,而脉冲整形技术又不能有效利用激励激光功率,通过理论分析非线性光谱压缩机理和总结已报导的实验方案,开发了一套灵活、紧凑的非线性光谱压缩装置,该装置可将宽带飞秒激光线宽压缩至几个波数,同时维持一半以上的激光功率,利用搭建的非线性光谱压缩装置所提供的高功率斯托克斯激励光源,分别在指纹区和静默区建立了两套光谱分辨率优于10 cm-1的高光谱SRS显微成像系统,同时在静默区系统中改进了光谱扫描装置,设计了一种基于检流计振镜的新型脉冲内部光谱扫描器。针对当前高光谱SRS成像主要用于动物细胞、组织和模型生物研究的现状,利用分子指纹振动,开展了高光谱SRS显微成像用于分析描绘木质素化学成分分布的研究。以拟南芥作为模型,运用高光谱SRS显微成像观察对比转基因拟南芥与野生型拟南芥,确立了高光谱SRS显微成像定量区分木质素中不同化学组份并实时监测木质素化学成分变化的能力。将研究进一步拓展至长狗尾草和玉米秸秆,利用高光谱SRS显微成像观察维管束内纤维细胞,结合多元曲线分辨(multivariate curve resolution,MCR)分析,首次揭示了木质素中醇和醛两种不同组份在植物细胞壁内的渐变分布。最后,针对目前多元SRS成像光谱探测范围较窄的问题,提出了一种利用反向啁啾脉冲实现多元SRS显微成像的新方法,利用玻璃棒和光栅对分别对泵浦光和斯托克斯光引入正、负啁啾,建立了一套基于反向啁啾脉冲的多元SRS显微成像系统,同时通过对DMSO样品进行成像观察,验证了该方法的可行性。本文的研究成果推动了受激拉曼光谱显微成像技术的进步,丰富并拓展了其成像应用研究,对SRS显微成像的进一步发展具有重要的科学意义。
[Abstract]:Stimulated Raman scattering (stimulated Raman, scattering, SRS) microscopic imaging technology is a kind of label free imaging technology emerging, the imaging contrast from the molecular vibration characteristics, so it has chemical selectivity and excellent chemical specificity. In addition, due to the SRS imaging with high resolution, high sensitivity and non-invasive etc., in less than ten years of development has become an important research tool in the field of life science. Based on the stimulated Raman spectrum imaging hyperspectral SRS and multi SRS is in every pixel has a spectrum, so it can not only distinguish between different molecules having overlapping Raman spectrum the chemical can provide more abundant information, become the research frontier of the last two or three years. However, the development of the time limit, the stimulated Raman spectrum imaging research and Many aspects need to be improved, the application still needs to develop. In view of this, this paper using spectral method for nonlinear focusing and spectrum compression and pulse shaping combined to carry out high resolution Raman spectral imaging technology and the element analysis of chemical constituents of membrane potential detection and application of wood research. In theory, this paper uses nonlinear coupling wave equation produced in the process of SRS signal was derived. The expression of the SRS signal is presented. Analyze and compare the differences between SRS and CARS spectrum of different non resonant background. The effects of laser polarization on different depolarization Raman vibration modes of SRS signal intensity, and reveals the basic principle of polarization modulated imaging SRS noise in SRS imaging was discussed in detail. The problem of SNR and background, pointed out that the SRS microscopic imaging system should have shot noise limit detection in ideal condition The mechanism analysis ability. The spectral focusing method to achieve high spectral coherent Raman scattering microscopic imaging, and the use of glass rod into linear chirp is established based on the hyperspectral imaging system SRS spectrum focusing method, aiming at the existing method of Raman frequency shift in calibration and signal intensity calibration problems, gave specific solutions at the same time. In the aspect of application research, for the first time to discuss the feasibility of detecting markers of cell membrane potential by SRS signal. The erythrocyte ghost model, completed the vibration spectrum image of single natural cell membrane, confirmed the high sensitivity spectrum of SRS imaging detection of single cell membrane. The cell membrane ion composition controlled by internal and external change across the membrane at the same time using potential stimulated Raman imaging under different cell membrane potential, found SRS spectrum with the membrane potential change changed significantly, the results show that S RS imaging is expected to be used for label free detection of cell membrane potential. Considering spectral focusing method of spectral resolution is poor, it is difficult to meet the application of fingerprint imaging area, and pulse shaping technology cannot effectively use laser through theoretical analysis, experimental scheme of nonlinear spectral compression mechanism and the total nodes have been reported, developed a set of flexible, nonlinear spectroscopy compact compression device, the device can be compressed to several broadband femtosecond laser linewidth wavenumber, while maintaining the laser power more than half of the high power Stokes using nonlinear spectral structures compression devices provide excitation light source, high spectral SRS imaging system two sets of spectral resolution better than 10 cm-1 respectively based on fingerprint and silence at the same time zone, improved spectral scanning device in a silent zone system, based on the design of a new type of vibrating mirror galvanometer in optical pulse Spectrum scanner. In view of the current high spectral SRS imaging is mainly used in animal cells, tissues and biological research status model, using molecular fingerprint vibration, carried out SRS hyperspectral microscopic imaging for analyzing and describing the distribution of wood chemical components. Using Arabidopsis as a model, using SRS hyperspectral microscopic imaging observation of transgenic and wild type of Arabidopsis Arabidopsis, established a high spectral SRS imaging quantitative ability to distinguish between different chemical components of lignin and lignin real-time monitoring of chemical composition changes. The research is extended to long bristlegrass and maize straw, the observation of fiber cells in vascular bundle SRS hyperspectral imaging, combined with multivariate curve resolution (multivariate curve, resolution, MCR) analysis reveals the first element in wood alcohol and aldehyde two different components in the plant cell wall in gradient distribution. Finally, according to the The narrow range of problems of SRS imaging spectral detection, puts forward a new method of using reverse chirped pulse multiple SRS microscopic imaging, the use of glass rod and the grating of the pump and Stokes light into the positive and negative chirp is established based on the multi SRS imaging system at the same time through reverse chirped pulse. The imaging observation of the DMSO sample, verify the feasibility of the method. This thesis promotes stimulated Raman spectrum imaging technology, enrich and expand the application of imaging research has important scientific significance of SRS imaging in further development.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O437.3;O439

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