植物與人類的生活息息相關，不僅能夠乾淨的產生各種人類賴以生存的資源，對調節自然氣候或是水資源的保育都扮演著不可或缺的角色，故人類對植物學的知識建立是相當重要的。而在眾多的研究中，葉綠素螢光反應了相當程度的植物生理現象，但在早期對葉綠素螢光的研究上，往往需要透過化學層析的方式將葉綠素由搗碎的葉片內萃取出來，不僅嚴重的破壞了葉片內部的微結構，也降低了實驗環境與真實情況的符合程度。
隨著時代的演進，開始有科學家將光學顯微術引入了植物學的相關研究當中。本研究所採用的雙光子螢光顯微術，即是一套具優異光學切片能力、低破壞性之光學顯微技術，不需要對葉片進行樣本的處理即可進行葉綠素螢光的研究。以雙光子螢光顯微術的優勢為基礎，我們更進一步在影像系統架構中結合了光譜擷取系統，能在單次量測下分別記錄樣品的顯微影像及光譜訊號，解決了以往在光譜分析中，由於無法同時觀測到影像，故無法定確定螢光所發出位置的問題，並透過對十九種不同植物的影像及光譜分析，驗證了此一系統架構能作為植物研究上一套有利的量測及分析工具。

Plants are closely related to humans’ daily life. It not only creates various living resources in a clean way but also plays an important role in weather regulation and water preservation. Therefore, it is necessary for human to establish the knowledge of botany. In many researches, chlorophyll fluorescence was found to reflect some significant plant physiological phenomenon. However, in the early researches of chlorophyll fluorescence, chlorophyll should be extracted from mashed leaves by chemical chromatography. These procedures not only destroy the micro-structures of leaves but also change the natural environment of leaves.
With the evolution of techniques, optical microscopy began to be introduced in botany studies. In this research, two-photon fluorescence microscopy, which has optical sectioning power and low photodamages, is used to study the chlorophyll fluorescence without any sample preparation. Based on the advantages of two-photon fluorescence microscopy, a spectral detection system is further integrated with the imaging system to obtain the two-photon fluorescence image and the fluorescence spectrum in a single measurement. With the image information, it enables to make sure where the fluorescence comes from and realize the fluorescence distribution in the leaves. Through the analysis of images and spectra obtained from 19 different plants, this system is verified to be a useful tool for measurement and analysis in botany researches.

[1] http://en.wikipedia.org/wiki/Botany.
[2] S. Hell, "Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering," Topics in fluorescence spectroscopy, pp. 361-426, 2002.
[3] J. M. Anderson, "Changing concepts about the distribution of photosystems I and II between grana-appressed and stroma-exposed thylakoid membranes," Photosynthesis Research, vol. 73, pp. 157-164, 2002.
[4] J. E. Mullet, et al., "Chlorophyll proteins of photosystem I," Plant physiology, vol. 65, pp. 814-822, 1980.
[5] http://inspirationlaboratories.com/halloween-science-fluorescent-chlorophyll.
[6] http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html.
[7] J. B. Pawley, Handbook of biological confocal microscopy: Springer, 1995.
[8] http://www.uni-muenster.de/Physik.PI/DeCola/trcm.html.
[9] http://www.olympusfluoview.com/theory/confocalintro.html.
[10] W. Kaiser and C. Garrett, "Two-Photon Excitation in CaF_ {2}: Eu^{2+}," Physical Review Letters, vol. 7, p. 229, 1961.
[11] http://www.fmhs.auckland.ac.nz/sms/physiology/biophysics/technique-s.aspx.
[12] W. Denk, et al., "Two-photon laser scanning fluorescence microscopy," Science, vol. 248, pp. 73-76, 1990.
[13] T. Bridgwater, "Biomass for energy," Journal of the Science of Food and Agriculture, vol. 86, pp. 1755-1768, 2006.
[14] http://zh.wikipedia.org/wiki/File:Photosynthesis_Overview.png.
[15] A. A. Benson, et al., "The Path of Carbon in Photosynthesis, XV. Ribulose and Sedoheptulose," 1952.
[16] Z. Csintalan, et al., "Changes in laser-induced chlorophyll fluorescence ratio F690/F735 in the poikilochlorophyllous desiccation tolerant plant Xerophyta scabrida during desiccation," Journal of Plant Physiology, vol. 152, pp. 540-544, 1998.
[17] http://cpacesclass.blogspot.tw/2013/02/advanced-biology-light-reactions.html.
[18] J. P. Thornber, "Chlorophyll-proteins: light-harvesting and reaction center components of plants," Annual Review of Plant Physiology, vol. 26, pp. 127-158, 1975.
[19] R. J. Strasser and W. L. Butler, "Fluorescence emission spectra of Photosystem I, Photosystem II and the light-harvesting chlorophyll a b complex of higher plants," Biochimica et Biophysica Acta (BBA)-Bioenergetics, vol. 462, pp. 307-313, 1977.
[20] R. Pedrós, et al., "Chlorophyll fluorescence emission spectrum inside a leaf," Photochemical & Photobiological Sciences, vol. 7, pp. 498-502, 2008.
[21] C. A. Palmer, et al., Diffraction grating handbook: Newport Corporation Springfield, OH, 2005.
[22] Newport, "Model：6034 , Spectral Calibration Lamp, Hg (Ne), 18, ±5 mA, 500 Hrs Rated Life."
[23] A. Santner and M. Estelle, "Recent advances and emerging trends in plant hormone signalling," Nature, vol. 459, pp. 1071-1078, 2009.
[24] N. Subhash and C. Mohanan, "Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower," Remote sensing of environment, vol. 60, pp. 347-356, 1997.
[25] C. Buschmann, "Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves," Photosynthesis Research, vol. 92, pp. 261-271, 2007.
[26] M. Koizumi, et al., "Light Gradients and the Transverse Distribution of Chlorophyll Fluorescence in Mangrove andCamelliaLeaves," Annals of Botany, vol. 81, pp. 527-533, 1998.
[27] http://twinkle_toes_engineering.home.comcast.net/~twinkle_toes_engineering/photosynthesis.htm.
[28] A. A. Gitelson, et al., "Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements," Journal of Plant Physiology, vol. 152, pp. 283-296, 1998.
[29] C. Buschmann, "The characterization of the developing photosynthetic apparatus in greening barley leaves by means of (slow) fluorescence kinetic measurements," Photosynthesis, vol. 5, pp. 417-426, 1981.
[30] H. K. Lichtenthaler, et al., "The chlorophyll fluorescence ratio F690/F730 in leaves of different chlorophyll content," Photosynthesis Research, vol. 25, pp. 295-298, 1990.
[31] G. B. Cordón and M. G. Lagorio, "Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models," Photochemical & Photobiological Sciences, vol. 5, pp. 735-740, 2006.
[32] 李宜真、蘇子正, "雙光子顯微術於生物組織的應用," 物理雙月刊(二十四卷三期), 2001.
[33] H. Lichtenthaler, et al., "Detection of vegetation stress via a new high resolution fluorescence imaging system," Journal of Plant Physiology, vol. 148, pp. 599-612, 1996.
[34] T. Tomo, et al., "Replacement of chlorophyll with di-vinyl chlorophyll in the antenna and reaction center complexes of the cyanobacterium Synechocystis sp. PCC 6803: Characterization of spectral and photochemical properties," Biochimica et Biophysica Acta (BBA)-Bioenergetics, vol. 1787, pp. 191-200, 2009.

[35] C. Xu and W. W. Webb, "Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm," JOSA B, vol. 13, pp. 481-491, 1996.
[36] F. W. Billmeyer and M. Saltzman, Principles of color technology: J. Wiley & sons, 1981.
[37] http://en.wikipedia.org/wiki/Hue.
[38] Z. Sestak and P. Siffel, "Leaf-age related differences in chlorophyll fluorescence (Review)," Photosynthetica, vol. 33, 1997.