Visualization and quantification of Cd sorption to bacteria using confocal laser scanning microscopy and Cd-specific fluorescent probes

Understanding metal sorption to bacteria is important for developing bioremediation strategies, for determining the fate of contaminants in ground water systems, and for defining controls on microbial metabolic processes in the environment. X-ray absorption spectroscopy (XAS) has been the only technique available for quantifying and mapping metal sorption to bacteria on an individual cell scale, but the XAS approach is time consuming and expensive, and quantitative mapping of the distribution of metals in complex samples is difficult with XAS. The development of metal-specific fluorescent probes, in conjunction with confocal laser scanning microscopy (CLSM), represents a novel and more effective approach for visualizing and quantifying biosorption. In this study, we test and calibrate the use of a commercially available Cd- and Pb-specific probe, Leadmium Green (LG), to quantify the distribution of Cd in bacteria-bearing systems. We have developed a treatment technique that uses a combination of the stock LG molecule and a chemically de-esterified form of the LG molecule (DELG) which enables us to quantify bacterial sorbed Cd. In this approach, we correlate the green fluorescence from LG and DELG with the fluorescence at a different wavelength of SYTO 63 cell stain which enables us to visualize and quantify bacterial cell Cd sorption on both a community and individual cell level. This approach, for the first time, yields a means of easily quantifying and visualizing metal adsorption onto individual bacterial cells which allows us to directly measure the distribution of Cd in bacteria-bearing environmental systems. In this study, we report the results of a range of control experiments that demonstrate the efficacy of this approach. We demonstrate that our chemical de-esterification of LG results in 100% conversion of the LG to the DELG form that is fluorescently active, and that untreated LG is not fluorescently active until it enters the cell. We also demonstrate that calibration curves for samples containing cells, LG, DELG, SYTO 63, and Cd can be developed. Our tests also demonstrate that neither LG nor DELG cause significant changes in sorption of Cd to bacterial cells. We present results from experiments showing that Cd sorption to bacteria is highly heterogeneous between cells, a result which differs from the previously held assumption of homogeneous sorption. The results of this study yield new insights into metal adsorption behavior in bacteria-bearing systems and our approach represents a powerful tool for quantifying the distribution of metals in complex realistic environmental samples.