Effects of Ethanol, Formaldehyde, and Gentle Heat Fixation in Confocal Resonance Raman Microscopy of Purple NonSulfur Bacteria
Effects of Ethanol, Formaldehyde, and Gentle Heat Fixation in Confocal Resonance Raman Microscopy of Purple NonSulfur Bacteria
复制标题
乙醇、甲醛和温和加热固定对紫色非硫细菌共聚焦共振拉曼显微镜的影响
DOI：
10.1002/jemt.20889
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发表时间：
2011-02-01
影响因子：
2.5
通讯作者：
Meinhardt-Wollweber, Merve
中科院分区：
文献类型：
--
作者：
Kniggendorf, Ann-Kathrin;Gaul, Tobias William;Meinhardt-Wollweber, Merve
共振拉曼显微镜非常适合于检查活的细菌样品，而无需进一步的准备。因此，相对较少考虑其与常见固定方法的兼容性。然而，细胞样品的固定是微生物科学中非常重要的工具，允许将样品保存在特定条件下以供进一步检查、未来测量、运输或以后参考。我们研究了三种常见的固定剂-乙醇，甲醛溶液，和温和的热-对三个属细菌物种的共振拉曼光谱的影响，球形红细菌DSM 158（T），沼泽红细菌DSM 123（T）和红色红细菌DSM 467（T），在共聚焦拉曼显微镜中保持类胡萝卜素和血红素发色团。此外，我们分析了显微镜载玻片的聚-L-赖氨酸涂层，广泛用于安装生物和医学样品，对后续的本地和固定样品的共聚焦拉曼测量的效果。结果表明，乙醇是优选的甲醛作为固定剂，如果应用小于24小时，而热固定有一个强大的，有害的影响的共振拉曼光谱的细菌。甲醛固定优于固定时间超过24小时，但会导致信号强度的整体下降。聚-L-赖氨酸涂层对用乙醇或热固定的样品的拉曼光谱没有明显的影响，但它进一步降低了用甲醛固定的样品的光谱中的信号强度，特别是在较高的波数处。Microsc. Res. Tech. 74：177-183，2011. (C)2010 Wiley-Liss，Inc.
Resonance Raman microscopy is well suited to examine living bacterial samples without further preparation. Therefore, comparatively little thought has been given to its compatibility with common fixation methods. However, fixation of cell samples is a very important tool in the microbiological sciences, allowing the preservation of samples in a specific condition for further examination, future measurements, transport, or later reference. We examined the effects of three common fixatives-ethanol, formaldehyde solution, and gentle heat-on the resonant Raman spectrum of three generic bacteria species, Rhodobacter sphaeroides DSM 158(T), Rhodopseudomonas palustris DSM 123(T), and Rhodospirillum rubrum DSM 467(T), holding carotenoid-and heme-chromophores in confocal Raman microscopy. In addition, we analyzed the effect of poly-L-lysine coating of microscope slides, widely used for mounting biological and medical samples, on subsequent confocal Raman measurements of native and fixed samples. The results indicate that ethanol is preferable to formaldehyde as fixative if applied for less than 24 h, whereas heat fixation has a strong, detrimental effect on the resonant Raman spectrum of bacteria. Formaldehyde fixation excels at fixation times above 24 h, but causes an overall reduction in signal intensity. Poly-L-lysine coating has no discernable effect on the Raman spectra of samples fixed with ethanol or heat, but it further decreases the signal intensity, especially at higher wavenumbers, in the spectra of samples fixed with formaldehyde. Microsc. Res. Tech. 74: 177-183, 2011. (C) 2010 Wiley-Liss, Inc.