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New harmonic generation microscopy techniques based on focal volume modelling.

机译：基于焦点体积建模的新谐波生成显微镜技术。

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摘要

Nonlinear microscopy has become an indispensable tool in the study of biological systems. It includes many nonlinear contrast mechanisms, each sensitive to different biological structures. However, interpretation of the images generated in nonlinear microscopy is a complex matter due to factors such as the structural complexity of the sample, phase relationships between the excitation beam and the detected signal and the nonlinear interactions in the focal volume of the microscope.;This thesis contains a new theoretical and numerical framework that describes the focusing of an excitation beam in a nonlinear microscope, the nonlinear optical interactions with the material in the focal volume, and the resulting nonlinear optical signal in the far field. The framework is the first to include reflection and refraction of the excitation beam and nonlinear signals by an arbitrary number of interfaces in the focal volume, which is especially significant for the interpretation of third harmonic generation (THG). It also uses the chirp-z transform to speed up calculations by orders of magnitude compared to numerical integration techniques.;The framework is used to investigate second harmonic generation (SHG) by collagen. Focusing effects alter polarization-dependent SHG measurements of collagen properties compared to the plane wave approximation, and this is verified experimentally. Furthermore, a technique of imaging the far field SHG radiation from collagen fibres is proposed, which can be used to extract the orientation of collagen fibres unambiguously. The framework is then applied to analyze the influence of interfaces on THG. Reflection effects at interfaces significantly affect THG, which leads to the development of a new super-resolution THG imaging technique based on backward-propagating THG. This super-resolution technique is experimentally demonstrated by imaging surface profiles with tens of nanometers resolution, which is the first time that such resolution is obtained in coherent nonlinear microscopy. Therefore, this imaging technique shows promise to become an important tool in high-resolution imaging of (biological) samples.;The theoretical and numerical framework provides a foundation for future research on the origin of nonlinear microscopy signals. The new imaging techniques based on this framework have great potential in quantifying fibrillar structures and interfaces in biological samples.
机译：非线性显微镜已成为生物系统研究中必不可少的工具。它包括许多非线性对比机制，每个机制都对不同的生物结构敏感。然而，由于诸如样品的结构复杂性，激发光束与检测到的信号之间的相位关系以及显微镜焦距中的非线性相互作用等因素，对非线性显微镜中产生的图像的解释是一件复杂的事情。论文包含了一个新的理论和数值框架，该框架描述了在非线性显微镜中激发光束的聚焦，在焦点体积中与材料的非线性光学相互作用以及在远场中产生的非线性光学信号。该框架是第一个包含激发光束和非线性信号通过焦点体积中任意数量的界面的反射和折射的框架，这对于解释三次谐波（THG）尤其重要。与数值积分技术相比，它还使用chirp-z变换将计算速度提高了几个数量级。该框架用于研究胶原蛋白产生的二次谐波（SHG）。与平面波近似法相比，聚焦效应改变了胶原蛋白性质的偏振依赖性SHG测量值，这已通过实验进行了验证。此外，提出了一种对来自胶原纤维的远场SHG辐射进行成像的技术，该技术可用于明确地提取胶原纤维的取向。然后将该框架应用于分析接口对THG的影响。界面处的反射效应会严重影响THG，这导致了基于向后传播THG的超高分辨率THG成像技术的发展。通过以数十纳米分辨率成像表面轮廓，实验证明了这种超分辨率技术，这是在相干非线性显微镜中首次获得这种分辨率。因此，这种成像技术显示出有望成为高分辨率（生物）样品成像的重要工具。；理论和数值框架为非线性显微镜信号起源的进一步研究奠定了基础。基于此框架的新成像技术在定量生物样品中的原纤维结构和界面方面具有巨大潜力。

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  • 作者

    Sandkuijl, Daaf.;

  • 作者单位

    University of Toronto (Canada).;

  • 授予单位 University of Toronto (Canada).;
  • 学科 Biophysics.;Optics.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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