Probing the physiological state of phytoplankton at the single-cell level
Keywords:flow cytometry, phytoplankton, physiology, molecular probes, fluorescent dyes
Probing the physiological state of phytoplankton at the single-cell level provides valuable insight in ecological studies as well as in environmental monitoring of pollution or UV impacts. This paper reviews the recent progress in assessing the physiological state of phytoplankton with flow cytometry by inherent cell properties such as cell size and chlorophyll autofluorescence, specific fluorescent dyes, and newly developed molecular probes and enzyme substrates. It is reported how nitrogen and iron limitation as well as the effect of copper pollution could be derived from changes in cell inherent properties. Effects of Cu were also recorded by monitoring cell membrane potentials and esterase activity. Photosynthetic capacity of algae was assessed by changes in chlorophyll fluorescence with the electron transport inhibitor DCMU, by a cytometric adaptation of the pump-and-probe approach, and molecular probes for Rubisco. Antibodies were also applied to mark non-terminal stages in the cell DNA replication cycle, to detect non-proliferating cells, to assess DNA damage caused by UV-B radiation and to quantify diatom stickiness. Fluorescein diacetate proved useful to discriminate metabolically active from inactive cells and to reveal strategies of dark survival in algae. The activity of alkaline phosphatase was recorded by a new fluorigenic substrate ELF, and polyclonal antibodies against nitrate reductase (NR) provided measurements of the NR abundance. An outlook will show how recent developments in molecular probes might affect the future analysis of marine ecosystems and their communities.
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