Development of an Epi-Illuminating Fiber Optic Microprobe for Analysis of Chloroplast Specialization and Measurement of Chemical Parameters within Plant Tissue.

这项工作将开发一种外延照明光纤微探针，可用于分析生物组织微观部位的代谢事件。来自可调谐染料激光器的单色光将被耦合到光纤中，并被引导到另一端，那里有一个微小的尖端（直径5微米），可以插入生物目标组织中。该光将用于激发固有分子、人工荧光染料的荧光或监测电子载体和代谢中间体的吸光度变化。我们建议在植物中开发和测试该仪器，但原则上，外延照明探针可用于各种生物系统。具体的开发工作将指向：(1)光合作用电子载体不同吸光度变化的专门化分析；(2)开发可用于监测细胞和组织内代谢变化（如CO2浓度）的光纤化学传感器。这将通过将嵌入荧光染料的聚合物薄膜附着在探针尖端来实现，探针尖端会随着探针环境的变化而发生荧光变化。外延照明光纤探头将提供活体和活体组织内的实时信息。响应时间快（ns），探针将具有高空间分辨率，因此它将有可能测量光散射组织、细胞内微位点的事件，并检查少量（3-4）细胞器（如叶绿体）的生理特化。

This work will develop an epi-illumination fiber optic microprobe that could be used to analyze metabolic events at microscopic sites within biological tissues. Monochromatic light from a tunable dye laser will be coupled into an optical fiber and guided to the opposite end where there is a microscopic tip (5 um diameter) that can be inserted within a biological target tissue. The light will be used to stimulate fluorescence of intrinsic molecules, artificial fluorochromes or to monitor absorbance changes of electron carriers and metabolic intermediates. We propose to develop and test the instrument within plants but in principle, the epi-illumination probe could be used in a wide variety of biological systems. Specific developmental efforts will be directed toward (1) analysis of the specialization of different absorbance changes of photosynthetic electron carriers; (2) the development of fiber optic chemical senors that can be used to monitor metabolic changes, such as CO2 concentration, within cells and tissues. This will be pursued by attaching polymeric films with embedded fluorochromes to the probe tip that undergo changes in fluorescence in response to changes in the probe environment. The epi-illumination fiber optic probe will provide information both in vivo and in real time within living tissues. The response time is rapid (ns) and the probe will have high spatial resolution so that it will be possible to measure events at microsites within light-scattering tissues, cells and to examine the physiological specialization of small numbers (3-4) of organelles such as chloroplasts.