葉綠素螢光已被認為可有效作為高等植物研究應用，作為非侵入性的方法評斷植物生理狀況，本研究使用線掃描式雙光子螢光超光譜顯微系統取得葉片樣本的葉綠素螢光超光譜影像，超光譜影像具有一維光譜與三維空間資訊，可取得最厚樣本深度為葉片樣本表面往下120 μm。 本研究配合主成分分析法及低溫螢光光譜量測，取得光合作用中光系統I及光系統II之基底螢光光譜，並引入吸收光譜修正螢光再吸收效應，利用最小平方法擬合葉片樣本中葉肉細胞三維空間各個位置的螢光光譜。 經由驗證量測光譜與擬合光譜之誤差，本研究使用之光譜擬合分析法可用於葉綠素螢光光譜分析。 ;Chlorophyll fluorescence is considered as a non-invasive method to monitor the physiological state of plants and is capable to apply for higher plants researches. A home-built line scanning two-photon fluorescence hyperspectral microscopy is used to record the chlorophyll fluorescence hyperspectral images containing 3D spatial and 1D spectral information in this research. The hyperspectral images within 120 μm beneath the leaf surface can be recorded. In this research, the principle component analysis method and cryogenic fluorescence spectrum measurement are applied to analyze the spectrum bases of Photosystem I and Photosystem II. To correct the re-absorption effect in spectrum analysis, absorption spectrum of intact leaf is measured and used to build the fitting function. Using least squares method, the fluorescence spectra of mesophyll cells at various locations in three-dimensional are fitted and analyzed. Through the errors between the measured and fitted fluorescence spectra, the fitting method used in this research is verified to be applicable for analysis of chlorophyll fluorescence.
