本論文應用奈秒解析螢光生命週期技術，量測小球藻（Chlorella sp.）在不同強度與波長的激發光照射之下，其光合作用光保護機制中的非光化學焠滅（non-photochemical quenching, NPQ）反應路徑。此研究的目的是探討小球藻之最佳養殖條件，希望同時兼顧光保護機制以及提高其光合作用效率。
激發光強度不同的實驗中，葉綠素a螢光生命週期的量測值不一樣。較強激發光照射會造成較長的初始螢光生命週期，反之，較弱激發光照射則會獲得較短的初始螢光生命週期。因為當光強達到一定程度時，光合作用系統II （Photosystem II）會關閉部分反應中心，以避免過多的能量進入電子傳遞鏈而造成細胞受損；無法進入反應中心的能量便以螢光形式釋放。因此，並非提高光強就可以促使綠藻快速生長。由於光合作用系統II開啟狀態的反應中心越多，光合作用效率越佳，則本研究應用螢光生命週期量測，可以找到綠藻養殖時的最佳光強。
非光化學反應焠滅機制分為三種：qE (Energy-dependent quenching)、qT (State transition)、qI (Photo-inhibition)，在本論文中探討qE與qT機制對光合作用效率的影響。qT機制在弱光時啟動，其反應時間持續數十分鐘。qE機制需要高光強照射才開啟，並且不同波段的光源也會造成qE反應的差異；qE反應時間維持數秒至數分鐘。則本研究設計一套光源系統，可以分別激發qT與qE機制。本研究量測qT與qE啟動時，葉綠素a之螢光生命週期和螢光光強在數分鐘內的連續變化，並計算出光合作用的光化學反應效率。
本研究藉由量測光保護作用相闗的螢光生命週期，獲得光合作用效率的數值，希望實驗結果對於光合作用相闗研究有所貢獻，例如：糧食問題以及綠色能源。

In this thesis, I applied fluorescence lifetime technique to study the regulation of non-photochemistry quenching (NPQ) pathways in living Chlorella sp. cells. The goal is to find the best culture condition for Chlorella sp., so that the cells have balanced NPQ while having optimized photosynthesis efficiency.
When exposed to light of different intensity, the starting lifetime of chlorophyll a during fluorescence transient changed. The stronger the intensity, the longer the starting fluorescence lifetime. This is because illumination intensity above certain level causes photosystem II reaction centers to close. It is a photoprotection mechanism that prevents excess energy to damage cells. The excess energy is then released as fluorescence. While the ratio of open/closed reaction centers decides photosynthesis efficiency, fluorescence lifetime measurement provides a way to find the optimal light intensity for desired ratio of open/closed reaction centers.
Non-photochemistry quenching pathways include qE (Energy-dependent quenching), qT (State transition), and qI (Photo-inhibition). In this thesis, I discussed qE and qT mechanisms and their effects on photosynthesis efficiency. qT is activated under low light and lasts from seconds to minutes. qE is activated by stronger light, and responses differently as light wavelength varies; the reaction acts tens of minutes. I designed an illumination setup to stimulate either qE or qT response. I took continuous fluorescence lifetime measurement when either qE or qT was activated, and then calculated the corresponding photosynthesis efficiency.
I demonstrated the advantages of fluorescence lifetime technique in studying non-photochemical quenching pathways and photosynthesis efficiency. The results in this thesis might contribute to research field related to food crisis and green energy.

Content
摘要 i
ABSTRCT ii
誌謝 iv
Content v
List of Tables vii
List of Figures viii
Chapter 1 Introduction 1
1.1 Motivation and objective 1
1.2 Previous researches 2
1.2.1 Introduction of photosynthesis 2
1.2.2 Introduction of photosynthesis 3
1.2.3 Pulse amplitude modulation (PAM) fluorometry and PSMT curve of fluorescence induction 11
1.3 Applications 14
Chapter 2 theory and method 17
2.1 Measurement of Chlorophyll a fluorescence by FLMS 17
2.2 The frequency domain and homodyne based fluorescence lifetime measurement system 17
2.2.1 The frequency domain technology 18
2.2.2 The explanation of mathematical formula 18
2.2.3 The homodyne technology 19
2.2.4 Fourier transform 20
2.2.5 Analysis of polar plot 20
2.3 The introduction of MLB FLMS 21
2.3.1 The optical pathways of two different LED emitters 22
2.3.2 The introduction of FPGA 22
2.3.3 Verification of MLB FLMS 23
2.4 Preparation of algae sample for qT experiment 24
2.4.1 Results of NCBI blast for qT factors 24
2.4.2 Growth condition for inducing qT 25
2.4.3 Algae incubation 26
2.4.4 Centrifugation and sealing condensed algae in glass 28
2.5 Preparation of algae sample for qE experiment 28
2.5.1 Results of NCBI blast for qE factors 28
2.5.2 Growth condition for inducing qE 29
Chapter 3 Results of qT and qE experiment 31
3.1 Design of qT experiment 31
3.2 Results of qT experiment 31
3.3 Design of qE experiment 35
3.4 Results of qE experiment 36
3.4.1 Comparison of weak blue and red LED illumination 37
3.4.2 Comparison of strong blue and red LED illumination 40
3.4.3 Comparison of weak and strong blue LED illumination 44
3.4.4 Comparison of weak and strong red LED illumination 46
3.4.5 Comparison of white light and strong blue/red LED illumination 47
Chapter 4 Discussion 52
4.1 Discussion of qT experiment 52
4.2 Discussion of qE experiment 53
Chapter 5 Conclusion 58
References 60
Appendix 64
A1 Protein sequences of qT factors 64
A2 Protein sequences of qE factors 67

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