Quantifying Trace Elements in Individual Aquatic Protist Cells with a Synchrotron X-ray Fluorescence Microprobe

Abstract

The study of trace metal cycling by aquatic protists is limited by current analytical techniques. Standard "bulk" element analysis techniques that rely on physical separations to concentrate cells for analysis cannot separate cells from co-occurring detrital material or other cells of differing taxonomy or trophic function. Here we demonstrate the ability of a synchrotron-based X-ray fluorescence (SXRF) microprobe to quantify the elements Si, Mn, Fe, Ni, and Zn in individual aquatic protist cells. This technique distinguishes between different types of cells in an assemblage and between cells and other particulate matter. Under typical operating conditions, the minimum detection limits are 7.0 x 10(-16) mol microm(-2) for Si and between 5.0 x 10(-20) and 3.9 x 10(-19) mol microm(-2) for Mn, Fe, Ni, and Zn; this sensitivity is sufficient to detect these elements in cells from even the most pristine waters as demonstrated in phytoplankton cells collected from remote areas of the Southern Ocean. Replicate analyses of single cells produced variations of <5% for Si, Mn, Fe, and Zn and <10% for Ni. Comparative analyses of cultured phytoplankton cells generally show no significant differences in cellular metal concentrations measured with SXRF and standard bulk techniques (spectrophotometry and graphite furnace atomic absorption spectrometry). SXRF also produces two-dimensional maps of element distributions in cells, thereby providing information not available with other analytical approaches. This technique enables the accurate and precise measurement of trace metals in individual aquatic protists collected from natural environments.

Copper Uptake, Intracellular Localization, and Speciation in Marine Microalgae Measured by Synchrotron Radiation X-ray Fluorescence and Absorption Microspectroscopy

“…26,6 These published studies used a tropical isolate of C. closterium and lower initial cell densities for P. tricornutum and Tetraselmis sp. In contrast, Twining and co-workers 16 Copper is an essential element for the growth of microalgae and it is used as a cofactor in a number of enzymes and proteins, e.g. in plastocyanin (photosynthetic electron transport), cytochrome c oxidase (mitochondrial electron transport) and ascorbate oxidase (ascorbic acid oxidation and reduction).…”

“…10 In addition, SR-XRF is capable of elemental distribution mapping within individual cells at a resolution of 0.2-0.3 µm and has successfully been used to study the uptake of metals in mammalian cells. [12][13][14][15] Studies by Twinning and coauthors 16,17 and Diaz 18 also showed that microprobe SR-XRF was useful in determining the elemental composition and distribution of elements (Si, P, S, K, Ca, Mn, Fe, Co, Ni, Cu and Zn) in a range of marine microalgae isolated from oceanic waters and in two freshwater algae in laboratory culture media, respectively. This study uses correlative microprobe SR-XRF and XANES to investigate the internalisation of Cu in Cu-exposed marine microalgal cells to determine the toxicological modes of action of the metal.…”

Speciation and Quantitative Mapping of Metal Species in Microbial Biofilms Using Scanning Transmission X-ray Microscopy

“…On the basis of the statistical precision of single pixel spectra (Figure S4), the detection limit in the present measurements was estimated to be ∼10 -17 mole µm -3 Fe or ∼50 g (∼1 mole) Fe/kg dried weight biofilm. This can be compared to a detection limit of 10 -20 mole µm -3 or ∼50 mg (∼1 mmol) Fe/kg dried weight biofilm for spatially resolved X-ray excited, X-ray fluorescence (XRF) cited for Fe in single cells (44). As outlined in the Supporting Information, we estimate that a detection limit in STXM similar to that of XRF could be obtained with longer dwell times and by combining signals from potentially widely spaced pixels based on similarity of their spectra.…”

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

“…These techniques have been applied in marine carbon, iron, and phosphorus studies, and several studies in the materials, atmospheric, biological, and earth sciences (Hitchcock and Ishii 1987;Boese et al 1997;Flynn et al 1998;Bertsch and Hunter 2001;Peak et al 2002;Twining et al 2003;Brandes et al 2004;Twining et al 2004;Lam et al 2006;Diaz et al 2008). Here, we present X-ray spectromicroscopic data from the examination of two algal species, Chlamydomonas sp.…”

“…High resolution examination of phosphorus content and speciation within minimally prepared particulate samples has become feasible with the recent advent of synchrotron based X-ray technologies (Myneni 2002;Twining et al 2003;Twining et al 2004;Brandes et al 2007). X-ray spectromicroscopy (combined X-ray spectroscopy and microscopy) is used to map elemental distributions at high spatial resolution and to determine submicron-scale chemical speciation in minimally treated particulate specimens, including complete individual cells.…”