Development of Saturation Pulse Fluorescence Imaging System and Its Applications to Plants under Light-Heat Stress

本研究開發了飽和脈衝螢光影像系統，以進行植物光熱逆境之生理狀態分析。系統中的光平面系統是藉由可程式邏輯控制器，依飽和脈衝葉綠素螢光量測方式所需要的弱調控光、光化學能光及飽和脈衝光三種不同強度光源，進行時間序列之控制。 影像擷取平台則搭配不同螢光激發狀態，進行攝影機訊號增益倍率之調控，以解決攝影機動態範圍之能力不足問題，再藉由影像擷取頻率之自動調變，增加螢光劇烈變化階段之時間解析能力，以及減少在螢光反應趨於穩定時之取樣速度；並於取像時一併記錄擷取影像之時間，再以自行開發之螢光影像處理程序，分析完整之螢光影像資訊。 當植物處於正常狀態和不同光熱逆境階段時，經由本系統分析所得之『葉綠素螢光非光化學淬熄指標』、『葉綠素螢光非光化學淬熄指標變化曲線』和『螢光指標強度影像』等三種螢光指標，相較於市售MINI-PAM之量測結果，驗證本系統具備檢測植物逆境生理變化之能力。 此外，因本系統可以獲得完整葉面之螢光反應，考慮葉面上各部位因逆境所產生之變異，與市售單點儀器比較後，本系統量測逆境植物有更為一致之量測結果。飽和脈衝葉綠素螢光影像系統提供另一種途徑，可讓研究者以非破壞性的方式探討植物生理現象。In this article, the saturation pulse fluorescence imaging system (SPFIS) was developed to analyze to physical status of plants under light and heat stress. One part of SPFIS, the light plane system, was controlled by a programmable logic controller (PLC) to supply three different lighting conditions, including weak modulated measuring beam, continuous actinic light and saturating pulse lights, during measuring period. The other part of SPFIS, fluorescence imaging system, was developed to grab images with different signal gain setting of the CCD camera according to the different lighting conditions. The function of gain setting was used to overcome the insufficiency of camera dynamic range. Besides, the imaging frequency of SPFIS was auto adjusted to increase the time resolution when the fluorescence reaction change quickly and to decrease the sampling rate when the fluorescence reaction is stable. The imaging time was also recorded. The serial fluorescence images and the time record of plants were computed by the designed image processes to extract the complete fluorescence image information. The plants, under normal and different phases of light and heat stress, were analyzed by SPFIS to obtain three fluorescence results, including non-photochemical quenching of chlorophyll fluorescence, non-photochemical quenching curve of chlorophyll fluorescence, and intensity image of fluorescence index. Compared with the measurement results of the same plant by mini-PAM, SPFIS had the ability to detect the variation of physical status when plants were under stress. Moreover, according to SPFIS could obtain the complete fluorescence reaction of full leaves and consider the variations of full leaves under stress, the measurement results showed more consistent than mini-PAM, when plants were under stress condition. Saturation pulse fluorescence imaging system provides another way for research workers to monitoring plant physiological in non-destroy style.誌 謝 I 摘 要 II ABSTRACT III 目 錄 I 圖 目 錄 III 表 目 錄 V 第一章 前言 1 1-1 前言 1 1-2 研究目的 3 第二章 文獻探討 4 2-1環境與植物 5 2-1-1植物與光線 5 2-1-2人工光源 8 2-2光合作用 9 2-3葉綠素螢光 11 2-3-1葉綠素螢光生成原理 11 2-3-2葉綠素螢光檢測技術發展 13 2-3-3飽和脈衝法 17 2-4葉綠素螢光影像 24 第三章 材料與方法 30 3-1飽和脈衝葉綠素螢光影像系統之建立 30 3-1-1系統設計概念 30 3-1-1-1激發光源及光源控制流程 30 3-1-1-2螢光影像擷取 32 3-1-2系統硬體開發 33 3-1-2-1硬體設備組件 33 3-1-2-2平面型激發光源系統 39 3-1-2-2影像擷取平台 43 3-1-3系統控制程式 44 3-1-3-1光源控制流程 44 3-1-3-2 影像擷取平台 47 3-1-3-2-1數位攝影機增益值自動調變 47 3-1-3-2-2數位攝影機取像頻率自動調變 54 3-1-4螢光影像擷取與分析 57 3-1-4-1影像擷取流程 57 3-1-4-2影像分析策略 58 3-1-4-3影像分析流程 59 3-1-5系統性能測試實驗 60 3-2試驗相關之輔助儀器設備 62 3-3實驗材料 66 3-4葉片螢光強度均勻性實驗 68 3-4-1實驗目的 68 3-4-2實驗設計 69 3-6 逆境植物量測實驗 71 3-6-1實驗目的 71 3-6-2實驗設計 72 第四章 結果與討論 75 4-1系統整合與架構 75 4-2系統性能 78 4-2-1平面型激發光源系統 78 4-2-2影像擷取平台 80 4-2-3螢光影像分析 82 4-3植物葉片各部位葉綠素螢光均勻性之探討 85 4-3-1正常狀態 85 4-3-2逆境狀態 89 4-3-3螢光指標強度影像 93 4-4逆境下植物螢光之表現 98 4-4-1螢光強度曲線變化 98 4-4-2螢光指標強度影像變化 105 第五章 結論與建議 120 參考文獻 12