| 摘要: | 葉綠素螢光已被許多研究證明可以有效作為觀察植物生長狀態的應用，其優勢在於能在不破壞葉子的結構下進行量測，且有較快的量測時間，然而目前多數的研究多以葉子整體的螢光訊號做量測，缺少了從葉子螢光訊號中各個螢光成分的角度去做分析，因此，本論文目的為架設一套頻率域螢光生命週期量測系統，並且結合多重螢光分析技術，量測不同植物在光合作用下，葉子中各個成分間的螢光訊號的變化。在本論文中，介紹了利用校正平面的方式解決實驗上所使用的光偵測器PMT 所造成電壓和相位的非線性效應，也透過螢光標準品證明本系統在量測螢光生命週期的正確性與精確性，在量測R6G 螢光標準品時，其理論的螢光生命週期為3.9 ns，實驗上量測調變深度和相位誤差率約為0.5%，而在量測Eosin Y 螢光標準品時，其理論的螢光生命週期為1.08 ns，實驗上量測調變深度誤差率約為12%，量測相位誤差率約為7.5%，推斷是量測時所使用的調制頻率較低，使系統對於量測較短的螢光生命週期樣本時存在較大的誤差。而在葉子螢光訊號的量測上，發現葉子整體的螢光生命週期的變化可分為反應區和穩態區，後續也嘗試將葉子的螢光生命週期變化透過極座標作分析，發現螢光生命週期在極座標上的分布可透過擬合的方式以一條直線來表示其變化，並且其螢光生命週期的變化是與葉子螢光強度存在關係。最後實驗中透過多重螢光分析技術分析觀察
葉子在光合作用下，葉子中各個螢光成分的強度和權重的消長的變化，同時結合螢光光譜的量測發現葉子螢光光譜中兩個特徵峰值(F685 與F730)的強度反應時間與螢光生命週期的反應時間十分接近，後續如能提升本系統對於多重螢光生命週期分析的正確性的話，相信能更明顯觀察出兩者間的關聯。;Chlorophyll fluorescence has been proved by many theses to be effective as an application for observing the growth state of plants. The advantage is that it can be measured without destroying the structure of the leaf, and it has a faster measurement time. However, most of the current studies measure the fluorescent signal of the leaf as a whole, and lack the analysis from the perspective of each fluorescent component in the fluorescent signal of the leaf. Therefore, the purpose of this thesis is to set up a frequency domain fluorescence lifetime measurement system, and combine multiple fluorescence analysis technology to measure the changes of the fluorescence signals of various components in the leaves of different plants under photosynthesis.In this thesis, using correction plane to solve the nonlinear effects of voltage and phase caused by the photodetector PMT used in the experiment. It also proves the correctness and accuracy of the system in measuring the fluorescence lifetime through fluorescent standards sample. When measuring the R6G fluorescent standard sample, its ideal fluorescence lifetime is 3.9 ns, and the error rate of modulation and phase is about 0.5%. When measuring Eosin Y fluorescence standard sample, its ideal fluorescence lifetime is 1.08 ns, and the error rate of modulation is about 12%, and phase is about 7.5%. It is inferred that the modulation frequency used in the measurement is too low, which makes the system have a large error when measuring samples with a short fluorescence lifetime.In the measurement of the leaf fluorescence signal, it was found that the change of the fluorescence lifetime of the leaf can be divided into the reaction zone and the steady zone. In the experiment also tried to analyze the changes of the fluorescence lifetime of leaves through polar coordinates. It was found that the distribution of the fluorescence lifetime on the polar coordinates can be represented by a straight line by fitting, and the change of the fluorescence lifetime is the same as that. Also, there is a relationship between leaf fluorescence intensity and the distribution of the fluorescence lifetime on the polar coordinates. In the final experiment, the multiple fluorescence analysis technology was used to analyze and observe the changes of the intensity and weight of each fluorescent component in the leaves under photosynthesis. At the same time, combined with the measurement of the fluorescence spectrum, two characteristic peaks (F685 and F730) in the fluorescence spectrum of the leaves were found. The response time of fluorescence spectral intensity is very close to the response time of the fluorescence lifetime. If the system can improve the accuracy of the multiple fluorescence lifetime analysis in the future, the correlation between the two can be more clearly observed. |
