基于共聚焦显微拉曼的真菌菌丝中几丁质的原位检测研究

In Vivo Study of Chitin in Fungal Hyphae Based on Confocal Raman Microscopy

几丁质是真菌细胞壁中一种重要的结构多糖,本文首次采用共聚焦显微拉曼技术对山茶刺盘孢菌的气生菌丝进行原位检测研究,首先确定了采集菌丝拉曼光谱的最优实验参数,并获得了菌丝,几丁质标准品和背景三种物质的典型拉曼光谱,对其中的特征峰进行归属分析,发现菌丝光谱中有明显的几丁质特征峰。然后对置于载玻片上菌丝的感兴趣区域进行拉曼光谱面扫描,通过主成分分析法发现面扫描区域中,菌丝和背景两种信号可以明显区分开来,结合主成分的载荷因子图得到了菌丝的两个主要的特征差异峰1 622和1 368cm^(-1),1 622cm^(-1)属于菌丝中几丁质的特征峰,而1 368cm^(-1)是来源于菌丝中的果胶多糖。最后通过对几丁质在1 622cm^(-1)特征峰波段附近范围积分,绘制了几丁质在菌丝中二维和三维的化学成像图,直观且无损的再现了几丁质在菌丝中的空间分布。

Chitin is an important structural polysaccharide of fungal cell wall.In this paper,aerial hyphae of Colletotrichum camelliae Massee was first studied by confocal Raman microscopy in vivo.Firstly,the optimal experimental parameters of hyphae for collecting the Raman spectra were determined,and the typical Raman spectra of hyphae,chitin standard and background were acquired.By comparing analysis,characteristic peaks of chitin were found in hyphae.Then,a region of interesting on hyphae was selected for Raman scanning.Through principal component analysis,the Raman signal of hyphae and background in the scanning area can be separated clearly.Combined with loading weight plot,two main characteristic peaks of hyphae were obtained,1 622cm^-1 was belong to chitin and 1 368cm^-1 was assigned to pectic polysaccharide.Finally,two and three dimension chemical images of fungal hyphae were realized based on Raman fingerprint spectra of chitin in a nondestructive way.