In the field of ocean biogeochemistry, one of the primary questions is how biological processes influence the chemical profile of the environment. Protists play a critical role in the transformation of biologically active elements in natural ecosystems. For example, the entry of elements into food webs is often mediated by autotrophic protists, and the return of elements to the abiotic world involves the action of mixotrophic and heterotrophic protists. To be able to predict more effectively the impact of changes to global geochemical cycles, an improved understanding of the biological mechanisms driving these mechanisms is necessary. Towards this end, novel approaches for the elemental composition of protists at the single-cell level are finding increased application.

Previous techniques for elemental analysis have largely focused on the bulk analysis of size-fractionated particulate material. However, this approach obscures the unique biogeochemical roles of various groups of marine microorganisms such as protists, diatoms, and diazotrophs. Recent progress in the elemental analysis of single cells - using electron, proton, and synchrotron X-ray microprobes - indicates the utility of these approaches in revealing the interactions between different functional groups with their environments.

In a recent review in the Journal of Eukaryotic Microbiology, Benjamin Twining - of the University of South Carolina, together with colleagues from Stony Brook University and Argonne National Laboratory - explores the capabilities and limitations of currently available techniques for single-cell elemental analysis. The authors also examine how the application of these methods to the analysis of cells collected from natural communities is enabling the accurate determination of cellular element stoichiometries - a critical factor in achieving a better understanding of elemental interactions and their role in global biogeochemical cycles.

Several microprobe approaches are applicable for single-cell elemental analysis, including (1) light microscopy in combination with fluorescent dyes, (2) electron energy-loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM), (3) analytical electron microprobe, via X-ray microanalysis (XRMA) and electron probe X-ray microanalysis (EPXMA), (4) proton microprobes, and (5) synchrotron X-ray microprobes. Of these techniques, the latter method offers the most advantages, including very high sensitivity, low background, and the ability to simultaneously detect more than ten elements. In addition, it enables the selective excitation of analytes, and offers the possibility of m-XANES (micro X-ray absorption near-edge structure) for chemical state mapping. The primary drawback of synchrotron X-ray microprobe analysis is that it is relatively slow.

Focusing on several recent studies of plankton biogeochemistry in low-iron waters of the Southern Ocean, Twining and colleagues describe how numerous insights have been revealed via single-cell analyses – including, for example, the discovery of significant inter-taxa differences in phosphorus, iron, and nickel quotas. In addition, appreciably different responses to iron fertilization were found between autotrophs and heterotrophs. Using two-dimensional sub-cellular mapping, the importance of iron to photosynthetic machinery, and of zinc to nuclear organelles, were also determined. Obtained using single-cell elemental analysis, variations seen in diatom silicification and cytoplasm content after iron fertilization are proving useful useful in elucidating the relationship between iron availability and silicification.

While no individual microprobe method is perfect all single-cell applications, each offers appreciable utility for the elucidation of plankton elemental composition. In time, these techniques will contribute significantly to our understanding of the role of ocean protists in global biogeochemical cycles.

Article by Twining et al. on single-cell elemental analysis, Journal of Eukaryotic Microbiology, May-June 2008

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