Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition
Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition
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DOI：
10.1111/j.1574-695x.2012.00956.x
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发表时间：
2012-07-01
影响因子：
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通讯作者：
Bjarnsholt, Thomas
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文献类型：
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作者：
Alhede, Morten;Qvortrup, Klaus;Bjarnsholt, Thomas
利用激光共聚焦扫描显微镜（CLSM）和扫描电镜（SEM）等多种显微技术对细菌生物膜进行成像。CLSM的一个限制是它的放大倍率有限，这是通过使用扫描电镜来解决的，扫描电镜提供了单个细菌如何定位和在生物膜内相互作用的高倍空间图像。然而，传统的扫描电镜受到制备过程中样品脱水要求的限制。由于生物膜主要由水组成，样品脱水可能会改变其形态。高放大而真实的图像对于理解生物膜的生理是很重要的。我们比较了传统的扫描电镜、聚焦离子束扫描电镜（FIB）和CLSM与不需要脱水的扫描电镜技术[低温扫描电镜和环境扫描电镜（ESEM）]。在冷冻扫描电镜的情况下，生物膜不脱水，但保持冷冻，以获得更接近样品的自然状态的高放大图像。使用ESEM技术，不需要任何准备。将这些方法应用到铜绿假单胞菌的生物膜上，我们发现生物膜的脱水对其外观有很大的影响，综合使用这些方法可以得到更真实的生物膜图像。
Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.