New applications of a biogenic silica deposition fluorophore in the study of oceanic diatoms

Silicon (Si) availability is known to be one of the main factors controlling the productivity and distribution of diatoms (Dugdale et al. 1995), but current chemical and microscopic methods do not allow discrimination of the key species responsible for Si biomineralization in mixed natural diatom assemblages. In 2001, new insights into biological silicification became available when studies with diatom cultures established that PDMPO [2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy] phenyl} oxazole] selectively binds to polymerizing silica and emits an intense fluorescence under ultraviolet (UV) excitation wherever newly formed Si is deposited (Shimizu et al. 2001). Here, we focus on adapting the PDMPO method to the study of Si use in natural diatom communities, including identification of individual cells carrying out new Si deposition. Experiments determined a simple and reproducible way to label actively silicifying diatoms, to preserve stained samples and identified the best optical tools to visualize the fluorescence properties of Si-bound PDMPO. The application of confocal multiphoton microscopy to the PDMPO method revealed unprecedented high-resolution three-dimensional (3D) imaging of new Si deposition within diatom cells, which can be used to study biogenic silica deposition in relation to the cell cycle. The quantitative aspects of this method were further explored and resulted in a protocol allowing simultaneous measurement of newly deposited biogenic silica and PDMPO incorporation in the same sample. This aspect was successfully tested on both cultures and field samples. Toxicity assays were also run on a mixed natural diatom community to ensure that the probe presented no deleterious effect on diatom growth, biogenic silica deposition, or silicic acid uptake rates over 24 h incubations and at the recommended concentration. There is also great potential for a better understanding of the biogeochemistry of Si in diatoms through coupling of this method to other quantifying tools, such as UV flow cytometry and image analysis.