Evaluation of microscopic techniques (epifluorescence microscopy, CLSM, TPE-LSM) as a basis for the quantitative image analysis of activated sludge
Evaluation of microscopic techniques (epifluorescence microscopy, CLSM, TPE-LSM) as a basis for the quantitative image analysis of activated sludge
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DOI：
10.1016/j.watres.2004.10.009
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发表时间：
2005-01-01
期刊：
影响因子：
12.8
通讯作者：
Morgenroth, E
中科院分区：
文献类型：
--
作者：
Lopez, C;Pons, MN;Morgenroth, E
从落射荧光显微镜到共焦激光扫描显微镜（CLSM）和双光子激发激光扫描显微镜（TPE-LSM）与荧光染色相结合的显微技术可以帮助评估复杂的微生物聚集体，例如活性污泥絮凝物。为了确定这些微观技术的应用限制，使用荧光活力指示剂（Baclight 细菌活力试剂盒，分子探针）染色后对来自三个不同来源的活性污泥样品进行了评估。开发了图像分析程序来量化红色和绿色染色细胞的总量、絮凝物内染色细胞的位置以及簇和丝中的空间组织。研究发现，合适的显微技术的选择很大程度上取决于所分析的微生物聚集体的类型。对于细胞密度高的絮凝物，优选使用 TPE-LSM，因为它可以提供聚集体内部结构的更清晰图像。落射荧光显微镜无法可靠地量化致密聚集体中的红色染色细胞。 CLSM 无法充分成像致密絮凝物内的内部丝状结构和染色细胞的位置。然而，对于典型的活性污泥絮凝物来说，落射荧光和 CLSM 被证明是足够的。 (C) 2004 Elsevier Ltd. 保留所有权利。
Microscopic techniques ranging from epifluorescence microscopy to confocal laser scanning microscopy (CLSM) and two photon excitation laser scanning microscopy (TPE-LSM) combined with fluorescent stains can help to evaluate complex microbial aggregates such as activated sludge floes. To determine the application limits of these microscopic techniques, activated sludge samples from three different sources were evaluated after staining with a fluorescent viability indicator (Baclight Bacterial Viability Kit, Molecular Probes). Image analysis routines were developed to quantify overall amounts of red and green stained cells, location of stained cells within the flocs, and the spatial organization in clusters and filaments. It was found that the selection of the appropriate microscopic technique depends strongly on the type of microbial aggregates being analyzed. For floes with high cell density, the use of TPE-LSM is preferred, since it provides a clearer image of the internal structure of the aggregate. Epifluorescence microscopy did not allow to reliably quantify red stained cells in dense aggregates. CLSM did not adequately image the internal filamentous structure and the location of stained cells within dense floes. However, for typical activated sludge floes epifluorescence and CLSM proved adequate. (C) 2004 Elsevier Ltd. All rights reserved.