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Developing a time-dynamic, label-free assay for apoptosis based on optical Gabor filtering.

机译：开发基于光学Gabor滤波的时间动态无标记凋亡检测方法。

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摘要

Apoptosis is a fundamental process in the homeostasis and development of mammals, dysfunction of its regulation is implicated in human disease, and it is of growing interest as a possible therapeutic target (especially in cancer). An apoptosis assay that is sensitive, quantitative, and label-free would therefore be useful in cancer drug discovery by offering a direct, time-dynamic in-situ assessment of apoptotic response to drug candidates.;This thesis documents the development of optical Gabor filtering into an apoptosis assay. Optical Gabor filtering was developed as an improvement to optical scatter imaging (OSI) capable of optical scatter-based detection of object size and aspect ratio without the need for a predictive scatter model or extrinsic labeling. Two versions of the optical Gabor filtering instrument (LCD-based and DMD-based). By applying a series of Gabor filters with varying spatial frequencies and orientations, a series of filtered images can be generated and processed to extract morphological parameters of interest. The parameters of size, orientedness and aspect ratio are developed and tested using polystyrene microspheres, marine diatoms and collagen gelation, respectively. Orientedness and aspect ratio are employed as morphometric parameters to study dynamic morphological alterations in apoptosis.;Aspect ratio measurements in apoptotic iBMK cells show a differential aspect ratio response to apoptosis induction by bax/bak expressing and bax/bak knockout cell variants. To determine the biological source of these variations, multimodal image acquisition of fluorescently-labeled mitochondria alongside Gabor filtered data is used in apoptotic endothelial cells. Results of this study show a drop in orientedness 1-2 h post-STS treatment in mitochondria-rich regions (reconnoitered by fluorescence) concomitant with mitochondrial fragmentation but absent elsewhere. This dynamic is similar but not identical to that observed from iBMK cells. OSI imaging of apoptotic BAEC also revealed that a previously reported drop in optical scatter image ratio in the first hour of apoptosis is also associated with mitochondria-rich regions. In the future, the differences between optical responses of BAEC and iBMK apoptotic cells will be elucidated with fluorescent tracking of mitochondria-rich areas in iBMK. Classification and other computational methods could also be employed to directly evaluate the optical Gabor-filtered data in the future.
机译：细胞凋亡是哺乳动物体内稳态和发育的基本过程，其调节功能障碍与人类疾病有关，作为一种可能的治疗靶点（尤其是在癌症中），其引起了越来越多的关注。因此，通过提供对候选药物凋亡反应的直接，时间动态的原位评估，一种灵敏，定量且无标记的凋亡检测方法将可用于癌症药物的发现。；本论文证明了光学Gabor过滤技术的发展。进入凋亡分析开发光学Gabor滤波是对光学散射成像（OSI）的一种改进，该技术能够基于光学散射的对象大小和纵横比检测，而无需预测散射模型或外部标记。两种版本的光学Gabor滤波仪器（基于LCD和基于DMD）。通过应用一系列具有变化的空间频率和方向的Gabor滤波器，可以生成并处理一系列滤波图像以提取感兴趣的形态参数。分别使用聚苯乙烯微球，海洋硅藻和胶原蛋白凝胶来开发和测试尺寸，取向度和纵横比的参数。方向性和长宽比被用作形态学参数，以研究凋亡中的动态形态学变化。；凋亡的iBMK细胞中的长宽比测量显示了bax / bak表达和bax / bak敲除细胞变异对凋亡诱导的不同长宽比响应。为了确定这些变异的生物学来源，在凋亡的内皮细胞中使用荧光标记的线粒体的多峰图像采集以及Gabor滤波的数据。这项研究的结果表明，STS处理后1-2小时，线粒体富集区域（通过荧光证实）的方向性下降，伴随线粒体破碎，而其他地方则没有。这种动态与从iBMK细胞观察到的动态相似但不完全相同。凋亡BAEC的OSI成像还显示，先前报道的凋亡第一小时光学散射图像比率下降也与线粒体富集区域有关。将来，通过荧光跟踪iBMK中线粒体富集区域，可以阐明BAEC和iBMK凋亡细胞的光学反应之间的差异。将来也可以使用分类和其他计算方法直接评估经过Gabor滤波的数据。

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  • 作者

    Pasternack, Robert M.;

  • 作者单位

    Rutgers The State University of New Jersey - New Brunswick and University of Medicine and Dentistry of New Jersey.;

  • 授予单位 Rutgers The State University of New Jersey - New Brunswick and University of Medicine and Dentistry of New Jersey.;
  • 学科 Engineering Biomedical.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 172 p.
  • 总页数 172
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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