Fast and Simple Tool for the Quantification of Biofilm-Embedded Cells Sub-Populations from Fluorescent Microscopic Images

Abstract

27Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro-and 28 eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell 29 counting by flow cytometry is often limited, for example when working with cells rigidly adhered 30 either to each other or to external surfaces like in bacterial biofilms or adherent cell lines and tissue 31samples. An alternative approach is provided by using fluorescent microscopy and confocal laser 32 scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a 33 given sample. To facilitate the quantitative assessment of cell fractions in microphotographs, we 34 suggest a simple two-step algorithm that combines the cell selection based and the statistical 35 approaches. Based on a series of experimental measurements performed on bacterial and eukaryotic 36 cells under various measurement conditions, we show explicitly that the suggested approach effectively 37 accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) 38 in all studied cases that are in good agreement with manual cell counting on microphotographs and 39 flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive 40 and user-friendly graphical interface for the initial adjustment of algorithm parameters to the 41 microscopic imaging conditions as well as for the sequential analysis of homogeneous series of similar 42 microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is 43 freely available online at

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