单细胞拉曼光谱结合多元统计方法分析不同酿酒酵母菌株的成分差异

Analysis of Components Difference of Yeast Strains Based on Laser Tweezers Raman Spectroscopy Combined with Multistatistical Analysis

应用单细胞拉曼光谱技术结合多元统计方法分析比较了六株不同来源的酿酒酵母菌株的同步化细胞,从而获知菌株间细胞成分的差异。利用连续密度梯度离心法分离酿酒酵母同步化细胞,分别对六个酵母菌株的同步化单细胞进行拉曼信号收集,并结合主成分分析(PCA)、辨别函数分析(DFA)两种多元统计分析光谱差异。结果表明,细胞的拉曼光谱表征其物质的基本组成与结构,六个菌株的拉曼光谱比较发现菌株间细胞的蛋白质、脂类的差异较大,而核酸类物质相差较小。六个菌株的拉曼光谱经多元统计分析可以在一定程度得到区分,辨别函数分析结果显示影响菌株间光谱差异最大共有14个峰:706,862,918,997,1 073,1 127,1 269,1 291,1 305,1 429,1 465,1 591,1 602和1 652cm-1,其中10个峰归属为蛋白质,3个峰归属为脂类,1个峰归属为核酸。根据DFA载荷,从1 340个谱线中提取出上述的14个谱峰进行新的DFA结果,与前面的结果是一致的。此方法能在单个活体细胞中进行,无需复杂前处理,能较好地研究遗传本质相差较小的酿酒酵母菌株间的成分差别。

The combination of laser tweezers and con-focusing Raman spectroscopy（LTRS） has made it possible to investigate single cells in aqueous media.In our experiments,a modified method of Percoll density gradient centrifugation was developed for isolating synchronous cells from six yeast strains and Raman spectra of single yeast cells were collected by LTRS as well,in which multiple statistical analysis,principal component analyses（PCA） and discriminant function analysis（DFA） were applied to distinguish synchronous cells between yeast strains statistically.The result showed that Raman spectra scattered from the trapping yeast cells could provide intrinsic molecular information,and there were remarkable difference among those of six yeast strains in Raman spectrogram which correspond to various biomacromolecule,the difference of protein as well as lipid were significantly higher than nucleinic acid between two of yeast strains randomly,and among the six strains,synchronized yeast cells can be discriminated using PCA and DFA based on 14 most contribution bands,706,862,918,997,1 073,1 127,1 269,1 291,1 305,1 429,1 465,1 591,1 602 and 1 652 cm-1,10 bands of which were from protein,3 bands were from lipoid,and 1 band was from nucleinic acid.To validate their significance,these variables were used to reperform DFA analysis and the re-plotted PC-DFA was the same as in the previous PC-DFA analysis,and the data would be considered validated.The authors show that the approach of laser tweezers Raman spectroscopy combined with multistatistical analysis has the potential to study difference among yeast strains.