Use of silica microspheres having refractive index similar to bacteria for conversion of flow cytometric forward light scatter into biovolume
Use of silica microspheres having refractive index similar to bacteria for conversion of flow cytometric forward light scatter into biovolume
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DOI：
10.1016/j.watres.2008.06.026
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发表时间：
2008-08-01
期刊：
影响因子：
12.8
通讯作者：
Ziglio, Giuliano
中科院分区：
文献类型：
--
作者：
Foladori, Paola;Quaranta, Alberto;Ziglio, Giuliano
本研究描述了一种快速转换流式细胞仪前向角光散射（FALS）到细菌生物体积的替代方法。利用Rayleigh-Gans理论解释了影响荧光强度的主要参数：考虑细菌细胞的参数特性和流式细胞仪的特性，对模型进行了灵敏度分析。对于粒径在细菌典型范围内的颗粒，其FALS强度主要受细菌细胞的体积和折射率的影响，并且与细胞的形状大致无关。所提出的从FALS强度到细菌生物体积的转换是基于通过使用具有尽可能类似于细菌的相对折射率的二氧化硅微球确定的校准曲线。该方法进行了验证，为两个不同的流式细胞仪（第一个配备了弧光灯，第二个激光）通过比较从FALS转换获得的生物体积分布与生物体积常规下测量落射荧光显微镜。从污水处理厂的细菌的具体情况进行了讨论。与耗时的传统显微镜方法相比，应用FALS测量细菌生物体积可能是一种非常有前途的工具，可以在几分钟内完成，同时在流式细胞术分析期间进行细菌计数。(C)2008爱思唯尔有限公司保留所有权利。
This research describes an alternative approach for the rapid conversion of flow cytometric Forward Angle Light Scattering (FALS) into bacterial biovolume. The Rayleigh-Gans theory was considered for explaining the main parameters affecting FALS intensity: sensitivity analysis of the model was carried out, taking into account the parameters characteristic of bacterial cells and characteristics of the flow cytometer. For particles with size in the typical range of bacteria, the FALS intensity is affected mainly by volume and refractive index of bacterial cells and is approximately independent of the shape of the cells. The proposed conversion from FALS intensity into bacterial biovolume is based on a calibration curve determined by using silica microspheres having relative refractive index as far as possible similar to that of bacteria. The approach was validated for two different flow cytometers (the first equipped with an arc lamp and the second with a laser) by comparing the biovolume distribution obtained from FALS conversion with the biovolume measured conventionally under epifluorescence microscopy. The specific case of bacteria taken from a WWTP was addressed. Compared to the time-consuming conventional microscopic approach, the application of FALS for sizing bacterial biovolume could be a very promising tool being completed in few minutes, simultaneously to the enumeration of bacteria during the flow cytometric analysis. (C) 2008 Elsevier Ltd. All rights reserved.