應用多波長奈秒螢光生命週期技術之光合作用中光保護分子機制檢測儀之系統開發

Abstract

藻類為未來生質能源的重要來源之一，而控制藻類培養環境，尤其是照光條件，可以有效提升其光合作用效率以及藻類生長速率。常見量測光合作用效率的方式，是利用葉綠體中的葉綠素a螢光強弱，判斷光合作用的狀態，並藉此找到最佳的藻類培養條件。然而，對於光合作用這類複雜系統，螢光的光強訊號往往難以定量，更無法區分出不同生化反應路徑所產出的訊號。本研究應用發光二極體，開發一套多波長奈秒解析螢光生命週期量測系統，應用於量測藻類葉綠素a的螢光生命週期訊號。本研究的目的包含：利用不同波長之量測光，區分不同色素分子所對應的光合作用反應路徑；同時，應用螢光生命週期搭配螢光光強訊號，解析各個反應路徑之生化狀況。本論文中使用的多色發光二極體，搭配自行設計的發光二極體驅動電路，此螢光生命週期量測架構可以有效提高發光二極體的射頻調變深度以及調變頻率，成功解析奈秒等級的螢光生命週期訊號。為了快速切換多波長激發光，在論文中自行設計發光二極體之切換電路。此研究中，量測不同光強但相同波長之激發光、以及相同光強但不同波長激發光的照明之下，活體藻類細胞的葉綠素a螢光生命週期變化，及葉綠素a螢光動態曲線，並討論其相闗的光合作用光保護機制。

Green algae is one of the most important source of biomass and biofuel. It has been known that culture condition, especially illumination light, decides photosynthesis efficiency hence the yield of biomass in green algae. Conventional instruments to monitor photosynthesis activities measure Chlorophyll a fluorescence intensity. However, as a very complex system, it is difficult to quantitatively identify photosynthesis reactions based on Chlorophyll a fluorescence intensity solely. In this thesis, I developed a nanosecond-resolved multi-wavelength fluorescence lifetime system, and applied this fluorescence lifetime system to study photosynthesis activities of green algae in vivo. In my lifetime system, the wavelength of illumination light excites different light-absorbing pigments in photosynthesis, therefore provides information on their corresponding pathways. Then, the state of each pathway can be evaluated by fluorescence lifetime in combination with fluorescence intensity. In my study, a multi-die light-emitting diode (LED) was used as the light source. The LED was modulated at radio frequency by signals from a field-programmable gate array (FPGA). I designed a switching circuit to address individual die on the multi-wavelength multi-die LED. This system was applied to measure Chlorophyll a signals of green algae in vivo. In the experiments, I changed either the wavelength or the intensity of activation light that excited Chlorophyll a, and I observed different fluorescence lifetime components and different dynamic decay curve of fluorescence intensity under different light condition. Related non-photochemical quenching pathways was discussed in this thesis.