Characterization of phosphorus, calcium, iron, and other elements in organisms at sub-micron resolution using X-ray fluorescence spectromicroscopy

Abstract

X-ray spectromicroscopy (combined X-ray spectroscopy and microscopy) is uniquely capable of determining sub-micron scale elemental content and chemical speciation in minimally-prepared particulate samples. The high spatial resolutions achievable with this technique have enabled the close examination of important microscale processes relevant to the cycling of biogeochemically important elements. Here, we demonstrate the value of X-ray microscopy to environmental and biological research by examining the phosphorus and metal chemistry of complete individual cells from the algal genera Chlamydomonas sp. and Chlorella sp. X-ray analysis revealed that both genera store substantial intracellular phosphorus as distinct, heterogeneously distributed granules whose X-ray fluorescence spectra are consistent with that of polyphosphate. Polyphosphate inclusions ranged in size from 0.3-1.4 μm in diameter and exhibited a nonspecies-specific average phosphorus concentration of 6.87 ± 1.86 μg cm-2, which was significantly higher than the average concentration of phosphorus measured in the total cell, at 3.14 ± 0.98 μg cm-2 (95% confidence). Polyphosphate was consistently associated with calcium and iron, exhibiting average P:cation molar ratios of 8.31 ± 2.00 and 108 ± 34, respectively (95% confidence). In some cells, polyphosphate was also associated with potassium, zinc, manganese, and titanium. Based on our results, X-ray spectromicroscopy can provide high-resolution elemental data on minimally prepared, unsectioned cells that are unattainable through alternative microscopic methods and conventional bulk chemical techniques currently available in many fields of marine chemistry.