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Alternative Illumination Beam Geometries in Fluorescence Microscopy for Clinical Prostate Pathology

机译:用于临床前列腺病理的荧光显微镜中的替代照明光束几何形状

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摘要

Light sheet microscopy, with the technique developed nearly a century ago and the first application toward fluorescence microscopy of biological specimens occurring in the late twentieth century, has seen a resurgence for applications requiring rapid or 3D imaging of biological samples. Conventional light sheet microscopy uses Gaussian beams that are the standard output of laser systems, and can be turned into a light sheet utilizing a cylindrical lens. However, highly focused Gaussian beams used in microscopy spread out quickly, leaving only a small section that can be imaged, and are also vulnerable to beam propagation distortion and steering in a scattering medium. In order to overcome the depth-of-focus versus resolution tradeoff inherent to Gaussian beams, non-diffracting beams have been proposed as solutions in applications where cellular resolution is required over a larger field-of-view (FOV). These non-diffracting beams, such as Bessel or Airy beams, offer significant improvements in depth-of-focus, but come with disadvantages, such as out-of-focus excitations that degrade contrast and image quality in fluorescent microscopy. However, due the interest in achieving higher FOVs without sacrificing resolution, there is a great deal of ongoing research looking at side lobe suppression techniques with non-diffracting beams.;In this thesis work we look at various methods of suppressing the side lobes of the Bessel beams and assess the contrast differences in comparison to a conventional Gaussian beam. Using a spatial light modulator and dual-axis microscope architecture, we create a test bed for beam shaping and comparing traits between the beam profiles. Differences in image contrast and signal-to-background ratio (SBR) are assessed when looking at fluorescent solutions, beads, and phantoms. In the final section, we look at preliminary experiments on using the sectioned Bessel beam with electronic confocal slit detection (eCSD) image processing and analyze the benefits, along with challenges faced when applying this technology to imaging cleared tissue specimens. Overall, the sectioned Bessel beam coupled with eCSD image processing allows us to achieve similar signal-to-background ratios (SBR) compared to a conventional Gaussian beam illumination scheme, while giving large improvements in depth-of-focus for a given resolution. Although we are unable to prove this in the current iteration of the microscope test bed, these properties are expected to be particularly useful in imaging cleared tissue samples.
机译:光片显微镜技术是近一个世纪前发展起来的技术,并且在二十世纪后期首次出现在生物样品的荧光显微镜上,这种技术在需要对生物样品进行快速或3D成像的应用中再次流行。常规的光片显微术使用高斯光束,该高斯光束是激光系统的标准输出,并且可以利用柱面透镜将其变成光片。但是,显微镜中使用的高度聚焦的高斯光束会迅速散开,仅留下一小部分可以成像,并且还容易受到光束传播畸变和散射介质操纵的影响。为了克服高斯光束固有的聚焦深度与分辨率的权衡,非衍射光束已被提出作为在较大视野(FOV)上需要蜂窝分辨率的应用中的解决方案。这些非衍射光束(例如贝塞尔光束或艾里光束)可显着改善焦深,但也有缺点,例如离焦激发会降低荧光显微镜的对比度和图像质量。但是,由于有兴趣在不牺牲分辨率的情况下获得较高的FOV,因此,正在进行大量研究,以研究非衍射光束的旁瓣抑制技术。在本论文中,我们研究了多种抑制旁瓣的方法。贝塞尔光束并评估与传统高斯光束相比的对比度差异。使用空间光调制器和双轴显微镜架构,我们创建了用于光束整形和比较光束轮廓之间特性的测试台。在查看荧光溶液,微珠和体模时,会评估图像对比度和信噪比(SBR)的差异。在最后一节中,我们将研究使用分段贝塞尔光束和电子共聚焦缝隙检测(eCSD)图像处理的初步实验,并分析其好处,以及将该技术应用于对清除的组织样本成像时面临的挑战。总体而言,与传统的高斯光束照明方案相比,分段的Bessel光束与eCSD图像处理相结合使我们能够实现相似的信噪比(SBR),同时在给定分辨率下可大大改善焦深。尽管我们无法在当前的显微镜测试床迭代中证明这一点,但是这些特性有望在对清除的组织样本成像中特别有用。

著录项

  • 作者

    Chia, Jeffrey.;

  • 作者单位

    The University of Arizona.;

  • 授予单位 The University of Arizona.;
  • 学科 Optics.;Biomedical engineering.;Medical imaging.
  • 学位 M.S.
  • 年度 2017
  • 页码 97 p.
  • 总页数 97
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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