開發可應用於葉綠素螢光分析的螢光生命週期量測系統

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：86

、訪客IP：3.147.126.146

姓名

曾皓柏(Hao-Po Tseng) 查詢紙本館藏

畢業系所

光電科學與工程學系

論文名稱

開發可應用於葉綠素螢光分析的螢光生命週期量測系統
(Development of a fluorescence lifetime measuring system for analyzing chlorophyll fluorescence)

相關論文

★ 非反掃描式平行接收之雙光子螢光超光譜顯微術	★ 以二次通過成像量測架構及降低誤差迭代演算法重建人眼之點擴散函數
★ LASER光源暨LED在老鼠毛生長的低能量光治療比較分析	★ 應用線狀結構照明提升雙光子顯微鏡解析度
★ 以同調結構照明顯微術進行散射樣本解析度之提升	★ 掃描式二倍頻結構照明顯微術
★ 小貓自泵相位共軛鏡於數位光學相位共軛與時間微分之研究	★ 鏡像輔助斷層掃描相位顯微鏡
★ 以數位全像術重建多波長環狀光束之研究	★ 相位共軛反射鏡用於散射介質中光學聚焦之研究
★ 雙光子螢光超光譜顯微術於多螢光生物樣本之研究	★ 倍頻非螢光基態耗損超解析之顯微成像方法
★ 葉綠素雙光子螢光超光譜影像於光合作用研究之應用	★ 雙光子掃描結構照明顯微術
★ 微投影光學切片超光譜顯微術	★ 使用結構照明顯微術觀察活體小鼠毛囊生長週期之變化

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 (2027-8-5以後開放)

摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 葉綠素螢光
★ 螢光生命週期

關鍵字(英)

論文目次

摘要 i
ABSTRACT ii
致謝 iv
目錄 v
表目錄 vii
圖目錄 ix
第一章緒論 1
1.1 研究動機 1
1.2 文獻回顧 2
1.3 螢光生命週期量測技術(FLIM)介紹 3
1.4 多重螢光生命週期量測方式比較 5
1.5 系統量測技術選用與目的 8
第二章實驗原理 9
2.1 螢光生命週期量測 9
2.2 極座標分析(Polar plot) 13
2.3 多重螢光生命週期原理與偉伯演算法(Weber algorithm) 14
2.4 光合作用與葉綠素螢光探討 17
第三章實驗架構 20
3.1 螢光生命週期量測系統架構 20
3.2 DAC 訊號控制方式 26
3.3 樣本製備 29
第四章實驗結果 31
4.1 螢光生命週期量測實驗過程 31
4.2 PMT 校正實驗設計與結果 33
4.3 螢光標準品系統驗證 38
4.4 不同濃度下螢光標準品量測結果與討論 44
4.5 多重螢光生命週期量測及結果分析 47
4.6 葉子螢光生命週期量測分析 53
第五章結論與展望 68
參考文獻 70

參考文獻

[1] L. P. Vernon, "Spectrophotometric determination of chlorophylls and pheophytins in plant extracts," Analytical Chemistry, vol. 32, no. 9, pp. 1144-1150, 1960.
[2] V. Petkova, I. D. Denev, D. Cholakov, and I. Porjazov, "Field screening for heat tolerant common bean cultivars (Phaseolus vulgaris L.) by measuring of chlorophyll fluorescence induction parameters," Scientia Horticulturae, vol. 111, no. 2, pp. 101-106, 2007.
[3] M. Havaux, "Temperature sensitivity of the photochemical function of photosynthesis in potato (Solanum tuberosum) and a cultivated Andean hybrid (Solanum x juzepczukii)," Journal of Plant Physiology, vol. 146, no. 1-2, pp. 47-53, 1995.
[4] D. Willits and M. Peet, "Measurement of chlorophyll fluorescence as a heat stress indicator in tomato: laboratory and greenhouse comparisons," Journal of the American Society for Horticultural Science, vol. 126, no. 2, pp. 188-194, 2001.
[5] V. Sinsawat, J. Leipner, P. Stamp, and Y. Fracheboud, "Effect of heat stress on the photosynthetic apparatus in maize (Zea mays L.) grown at control or high temperature," Environmental and Experimental Botany, vol. 52, no. 2, pp. 123-129, 2004.
[6] J. Gamon, J. Penuelas, and C. Field, "A narrow-waveband spectral index that tracks diurnal changes in photosynthetic efficiency," Remote Sensing of environment, vol. 41, no. 1, pp. 35-44, 1992.
[7] J. Peñuelas, I. Filella, and J. A. Gamon, "Assessment of photosynthetic radiation‐use efficiency with spectral reflectance," New Phytologist, vol. 131, no. 3, pp. 291-296, 1995.
[8] G. Schmuck et al., "Chlorophyll fluorescence lifetime determination of waterstressed C3-and C4-plants," Radiation and Environmental Biophysics, vol. 31, no. 2, pp. 141-151, 1992.
[9] 蘇柏宇, "葉綠素雙光子螢光超光譜影像於光合作用研究之應用," 碩士, 光電科學與工程學系, 國立中央大學, 桃園縣, 2016. [Online]. Available: https://hdl.handle.net/11296/8er84x
[10] F. Franck, P. Juneau, and R. Popovic, "Resolution of the photosystem I and photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature," Biochimica et Biophysica Acta (BBA)-Bioenergetics, vol. 1556, no. 2-3, pp. 239-246, 2002.
[11] S. Evain, J. Flexas, and I. Moya, "A new instrument for passive remote sensing: 2. Measurement of leaf and canopy reflectance changes at 531 nm and their relationship with photosynthesis and chlorophyll fluorescence," Remote Sensing of Environment, vol. 91, no. 2, pp. 175-185, 2004.
[12] R. Datta, T. M. Heaster, J. T. Sharick, A. A. Gillette, and M. C. Skala, "Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications," Journal of biomedical optics, vol. 25, no. 7, p. 071203, 2020.
[13] T. W. Gadella Jr, T. M. Jovin, and R. M. Clegg, "Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale," Biophysical chemistry, vol. 48, no. 2, pp. 221-239, 1993.
[14] A. Squire, P. J. Verveer, and P. Bastiaens, "Multiple frequency fluorescence lifetime imaging microscopy," Journal of Microscopy, vol. 197, no. Pt 2, pp. 136-149, 2000.
[15] G. Weber, "Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements," The Journal of Physical Chemistry, vol. 85, no. 8, pp. 949-953, 1981.
[16] S. Schlachter et al., "mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Optics Express, vol. 17, no. 3, pp. 1557-1570, 2009.
[17] R. E. Dalbey, J. Weiel, W. J. Perkins, and R. G. Yount, "Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry," Journal of biochemical and biophysical methods, vol. 9, no. 3, pp. 251-266, 1984.
[18] "A picture of fluorescence lifetime." https://iss.com/resources/research/technical_notes/K2CH_FLT.html (accessed.
[19] N. Boens et al., "Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy," Analytical chemistry, vol. 79, no. 5, pp. 2137-2149, 2007.
[20] J. B. Reece, M. R. Taylor, E. J. Simon, and J. L. Dickey, Campbell biology: concepts & connections. Benjamin Cummings San Francisco, CA, 2012.
[21] S. Matsubara, Y.-C. Chen, R. Caliandro, and R. M. Clegg, "Photosystem II fluorescence lifetime imaging in avocado leaves: contributions of the lutein-epoxide and violaxanthin cycles to fluorescence quenching," Journal of Photochemistry and Photobiology B: Biology, vol. 104, no. 1-2, pp. 271-284, 2011.
[22] C. Buschmann, "Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves," Photosynthesis Research, vol. 92, no. 2, pp. 261-271, 2007.
[23] "CMOS (C11440-22CU, Hamamatsu ) datasheet." https://wiki.umontreal.ca/download/attachments/189567146/Hamamatsu_Orca%20Flash%204.0_V2_C11440-22CU_Technical%20Note.pdf?version=1&modificationDate=1627866466000&api=v2 (accessed.
[24] "Data Sheet. AD9959. Rev. C." https://www.analog.com/media/en/technical-documentation/data-sheets/AD9959.pdf (accessed.
[25] A. Penzkofer and Y. Lu, "Fluorescence quenching of rhodamine 6G in methanol at high concentration," Chemical physics, vol. 103, no. 2-3, pp. 399-405, 1986.
[26] K. Selanger, J. Falnes, and T. Sikkeland, "Fluorescence lifetime studies of Rhodamine 6G in methanol," The Journal of Physical Chemistry, vol. 81, no. 20, pp. 1960-1963, 1977.
[27] E. Slyusareva and M. Gerasimova, "pH-dependence of the absorption and fluorescent properties of fluorone dyes in aqueous solutions," Russian Physics Journal, vol. 56, no. 12, pp. 1370-1377, 2014.
[28] M. A. Mastanduno, S. Jiang, R. DiFlorio-Alexander, B. W. Pogue, and K. D. Paulsen, "Automatic and robust calibration of optical detector arrays for biomedical diffuse optical spectroscopy," Biomedical optics express, vol. 3, no. 10, pp. 2339-2352, 2012.
[29] G. I. Redford and R. M. Clegg, "Polar plot representation for frequency-domain analysis of fluorescence lifetimes," Journal of fluorescence, vol. 15, no. 5, pp. 805-815, 2005.

指導教授

陳思妤

審核日期

2022-8-30

推文