Quantification of phosphorus in single cells using synchrotron X-ray fluorescence

Phosphorus abundance was quantified in individual phytoplankton cells by synchrotron X-ray fluorescence and compared with bulk spectrophotometric measurements to confirm accuracy of quantification. Figures of merit for P quantification on three different types of transmission electron microscopy grids are compared to assess possible interferences. Phosphorus is required for numerous cellular compounds and as a result can serve as a useful proxy for total cell biomass in studies of cell elemental composition. Single-cell analysis by synchrotron X-ray fluorescence (SXRF) enables quantitative and qualitative analyses of cell elemental composition with high elemental sensitivity. Element standards are required to convert measured X-ray fluorescence intensities into element concentrations, but few appropriate standards are available, particularly for the biologically important element P. Empirical P conversion factors derived from other elements contained in certified thin-film standards were used to quantify P in the model diatom Thalassiosira pseudonana, and the measured cell quotas were compared with those measured in bulk by spectrophotometry. The mean cellular P quotas quantified with SXRF for cells on Au, Ni and nylon grids using this approach were not significantly different from each other or from those measured spectrophotometrically. Inter-cell variability typical of cell populations was observed. Additionally, the grid substrates were compared for their suitability to P quantification based on the potential for spectral interferences with P. Nylon grids were found to have the lowest background concentrations and limits of detection for P, while background concentrations in Ni and Au grids were 1.8- and 6.3-fold higher. The advantages and disadvantages of each grid type for elemental analysis of individual phytoplankton cells are discussed