Methodologies in XRF Cytometry.

Abstract

The "inorganic physiology" of a cell – that is, the storage, uptake, efflux and regulation, of metal ions, is critical to understanding the role(s) that metal ions play in biology. Two new methods for cellular elemental analysis are developed. The first is the creation of an x-ray fluorescence flow cytometer that can determine the total elemental content of single cells. This instrument can directly measure population heterogeneity for metals in the μM to mM concentration range with fL sample volumes, a measurement that is difficult using most analytical methods. Bovine red blood cells (bRBCs) were found to have mean concentrations of ~100 μM Zn and ~15 mM Fe; NIH3T3 and yeast contained ~50 μM Zn and ~130 μM Zn, respectively. These data demonstrated that there is significant variability in the Zn and K content of NIH3T3 cells and in the Fe content of bRBCs. Fe content for bRBCs showed a 1.9-fold difference between the lowest and highest quartiles, variability that is dominated by biological variability and not experimental uncertainty. Likewise, NIH3T3 cells showed 2.3- and 2.8- fold differences between the 1st and 3rd quartiles for Zn and K, respectively. Second, fitting methods for x-ray fluorescence microprobe imaging were improved. A major advancement was the development of a blank subtraction method to correct the background and calculate elemental concentrations; this gives a significant improvement in quantitation. Comparison of the new method against the more commonly used baseline subtraction demonstrated not only better precision, but also improved instrument calibration. Differences in quantitation are biologically relevant. Additionally, blank subtraction allows superior sensitivity, best demonstrated with the detection of Cl. This method was used to image and determine the elemental content in NIH3T3 cells in the presence and absence of Cd, confirming 3-fold decrease in Zn content following Cd exposure.