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This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium  Shewanella oneidensis  MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

Surface-Enhanced Raman Imaging of Intracellular Bioreduction of Chromate in Shewanella oneidensis