三維雷利散射顯微術及成像於活體葉綠體中基粒之研究

Abstract

在此研究中，我們著重於雷利散射共聚焦顯微術及成像系統的應用，尤其在亞微米及奈米粒子的觀測。基粒 (Grana)為數個盤狀的類囊體堆疊形成亞微米的大小而散布於葉綠體裡，故可視為行光合作用的最小單位。基粒的大小與其化學組成被認為具有不均質性，從這觀點出發，結合顯微影像及光譜的量測有助於分子的辨別並討論基粒的大小與光合作用的關係。

從水蘊草細胞的共聚焦影像中，我們可以清楚的觀察到基粒隨機地在葉綠體裡頭分布。量測單一基粒的光散射光譜時，我們察覺每一個基粒的光譜都不完全相同，並且觀察到基粒的位置 (在葉綠體的中間或外圍，或是葉綠體坐落在葉子裡的任一位置)不影響光譜的量測結果。最重要的發現是基粒的尺寸相關性：當基粒的尺寸越大時，其波長會往紅外線區移動。此外，我們還用極化光測基粒的特性，證實它是光學各向異性 (optical anisotropy)。根據分析結果我們推測：在較大的基粒裡存有較多的葉綠素分子，此外，在雷射照射下，尺寸大的基粒裡分子排列得愈發整齊，這也暗示了基粒光合作用的效率與它的尺寸大小有關。

為證實實驗數據的可信度，我們製備了含有金奈米粒子的晶體，並且成功地測得金奈米粒子的光散射光譜，即便它非常小又存在於晶體之中。另一方面，此實驗亦證實了我們系統的優異性能。

In this study, we focus on the application of confocal Rayleigh scattering microspectroscopic and imaging system of submicron- to nanometer-sized particles, especially in vivo biological substances. In chloroplast of plant cell, submicronmeter-sized grana are widely arranged and play a role of light-harvesting. The size and chemical composition of grana are considered to possess inhomogeneity. With this viewpoint, combining confocal microscopic imaging with spectroscopic measurement helps us to identify the molecules and to discuss the function of the grana in terms of their sizes.

Confocal images of chloroplasts of Egaria densa depicted the spatial distribution of grana. The light scattering spectra of the grana depend on the granum size, although the spectra are independent on the position of the granum, in the center or on the exterior of the chloroplast. The greatest finding is that the spectrum is red-shifted as the granum size enlarges. Polarization measurement indicated that grana are optically anisotropic. According to our analyses, the obtained result suggested that there are more chlorophyll pigments in larger granum and the molecules align much better in larger ones when irradiated by laser. Based on the inference, we suppose that the light-harvesting property of grana is reflected in size.

The reliability of the results was supported by measurement of the light scattering spectra of 40 nm Au nanoparticles embedded in the glycine crystal, which can be considered as a model system containing nanoparticles inside local anisotropic surrounding, similar to that in the chloroplast. This model system also demonstrated that confocal Rayleigh scattering microspectroscopic and imaging system is a powerful tool for detection of single nanoparticles.