The goal of this work was to develop and demonstrate the utility of microscope‐based image cytometry (ICM) as a method for quantifying nuclear DNA content and cell cycle phase distribution in microalgae both in culture and in natural blooms, as an alternative to flow cytometry (FCM). To do so, aliquots from the same samples of the dinoflagellate Karlodinium veneficum were examined using both ICM and FCM. ICM specimen preparation and data acquisition methods were optimized to improve precision and agreement between the two techniques. Accuracy and precision of DNA measurements by ICM were significantly higher using the DNA fluorophore DAPI compared to SYBR® Green I. Milli‐Q H₂O was found to be superior to Tris‐EDTA as a staining and slide preparation solution for ICM analyses. Lower‐powered objective magnification (10x, 20x) in image acquisition for ICM produced higher precision in nuclear DNA measurements. Overall precision of ICM analysis of DAPI‐stained K. veneficum cells was comparable to FCM, with respective 1C DNA peak coefficients of variation as low as 6.2%. Cell cycle distributions of mid‐log culture samples analyzed by both ICM and FCM were in agreement (two‐way ANOVA; p = 0.93); while distributions analyzed in a field sample were similar but not identical (Z‐test; p < 0.001). Overall, the results show the feasibility of ICM as a useful tool for microalgal cell cycle analysis, with the potential for more flexible application to mixotrophic/phagotrophic species and complex field populations.

中文翻译：

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混合营养型鞭毛藻的定量核DNA含量和细胞周期分析的图像细胞计数法

这项工作的目的是开发和证明基于显微镜的图像细胞计数法（ICM）作为流式细胞术的一种替代方法，该方法可用于定量测定培养和自然繁殖过程中微藻中核DNA的含量和细胞周期的相位分布。 （FCM）。要做到这一点，从甲藻的同一样品等分Karlodinium veneficum同时使用ICM和FCM进行了检查。优化了ICM标本制备和数据采集方法，以提高两种技术之间的精确度和一致性。与SYBR®Green I相比，使用DNA荧光团DAPI通过ICM测量DNA的准确性和精密度要高得多。Milli-QH ₂O被认为是优于Tris-EDTA的用于ICM分析的染色和载玻片制备溶液。ICM图像采集中的低倍物镜放大倍率（10倍，20倍）在核DNA测量中产生了更高的精度。对DAPI染色的K.veneficum细胞进行ICM分析的总体精度可与FCM相媲美，其1C DNA峰变异系数分别低至6.2％。通过ICM和FCM分析的对数中期培养物样品的细胞周期分布是一致的（双向方差分析；p = 0.93）；实地样本中分析的分布相似但不完全相同（Z检验；p <0.001）。总体而言，结果表明ICM作为微藻细胞周期分析有用工具的可行性，并可能更灵活地应用于混合营养/吞噬营养物种和复杂的田间种群。