RESUMO
BACKGROUND: Active fluorescence techniques are becoming commonly used to monitor the state of the photosynthetic apparatus in natural populations of phytoplankton, but at present these are bulk water measurements that average all the fluorescent material in each sample. Here we describe two instruments that combine individual-cell "pump-during-probe" (PDP) measurements of chlorophyll (Chl) fluorescence induction, on the time scale of 30 to 100 micros, with flow cytometric or visual characterization of each cell. METHODS: In the PDP flow cytometer, we measure Chl fluorescence yield as a function of time during a 150 micros excitation flash provided by an argon ion laser; each particle is subsequently classified as in a conventional flow cytometer. In the PDP microfluorometer, individual cells in a sample chamber are visually identified, and fluorescence excitation is provided by a blue light-emitting diode that can be configured to provide a saturating flash and also a subsequent series of short flashlets. This sequence allows both saturation and relaxation kinetics to be monitored. RESULTS: Phytoplankton from natural samples and on-deck iron-enrichment incubation experiments in the Southern Ocean were examined with each PDP instrument, providing estimates of the potential quantum yield of photochemistry and the functional absorption cross section for photosystem 2, for either individuals (for cells larger than a few micrometers) or populations (for smaller cells). CONCLUSIONS: Results from initial field applications indicate that single-cell PDP measurements can be a powerful tool for investigating the nutritional state of phytoplankton cells and the regulation of phytoplankton growth in the sea.
