Calcein Staining as a Tool to Investigate Coccolithophore Calcification
Calcein Staining as a Tool to Investigate Coccolithophore Calcification
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DOI：
10.3389/fmars.2018.00326
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发表时间：
2018-09-18
影响因子：
3.7
通讯作者：
Taylor, Alison R.
中科院分区：
文献类型：
--
作者：
Fox, Emily;Meyer, Erin;Taylor, Alison R.
尽管球藻具有海洋学和地质学意义，但其胞内产生和随后分泌碳酸钙的细胞机制仍然知之甚少。为球石和球球贴上标签以跟踪其生产的工具将具有很大价值。因此，我们评估了荧光素的衍生物钙黄绿素作为一种荧光标记球虫的方法的使用。Calcein方法在不影响球石结构的情况下，很容易标记一系列球石生物体物种中先前存在的球虫，包括二倍体和单倍体生活史阶段。方解石染色通过荧光和共聚焦显微镜进行验证，染色和未染色的细胞和球虫很容易通过流式细胞术区分开来。染色的球虫的荧光被保留3天，使我们能够通过区分先前存在的球虫和接触钙黄绿素后产生的非荧光球虫的积累和分布来确认它们的极地分泌。在8天的时间里，钙黄绿素处理对光合作用生理、外部方解石形态或细胞生长速率没有显著影响。因此，钙黄绿素染色方法代表了一种简单的非侵入性、无毒的光学技术，可以‘标记’碳酸钙球石并跟踪它们的生产，以响应环境操纵或药物治疗。此外，球囊的钙调素染色允许在一组细胞中检测到钙化、生长和细胞分裂的不同模式。这是第一次使用钙黄绿素对钙化浮游植物的生物矿物结构进行染色，这种方法有可能应用于详细的细胞学研究以及对培养细胞或野外种群的高通量分析。
Despite the oceanographic and geological significance of coccolithophores, the cellular mechanisms that underlie the intracellular production and subsequent secretion of their CaCO3 coccoliths remain poorly understood. Tools for labeling coccoliths and coccospheres in order to track their production would be of great value. We therefore evaluated the use of calcein, a derivative of fluorescein, as a method to fluorescently label coccoliths. The calcein method readily labeled pre-existing coccospheres in a range of coccolithophore species, including diploid and haploid life history phases, without compromising the coccolith structure. Calcite staining was verified though epifluorescence and confocal microscopy, and both stained and unstained cells and coccoliths were readily distinguished using flow cytometry. The fluorescence of stained coccoliths was retained for > 3 days allowing us to confirm their polar secretion by distinguishing pre-existing coccoliths from the accumulation and distribution of non-fluorescent coccoliths produced after calcein exposure. The calcein treatment had no significant effect on photosynthetic physiology, external calcite morphology, or growth rates of the cells over an 8-day period. The calcein staining method therefore represents a simple non-invasive, non-toxic optical technique to 'tag' calcium carbonate coccoliths and track their production in response to environmental manipulations or pharmacological treatments. Moreover, calcein staining of the coccosphere allowed for heterogenous patterns of calcification, growth, and cell division to be detected in a population of cells. This is the first description of the use of calcein to stain the biomineral structures of calcifying phytoplankton and this approach has the potential to be applied to detailed cytological investigations as well as high-throughput analysis of cultured cells or field populations.