PNNL

Abstract

We report on the imaging of living bacterial cells by using a new correlated tapping-mode atomic force microscopy (AFM) and confocal al fluorescence lifetime imaging microscopy (FLIM). Different methods of preparing the bacterial sample were explored for optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells on poly-1-lysine coated surfaces and agarose gel coated surfaces. We have found that the agarose gel containing 99% buffer can provide a local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and single-to-noise ration of the AFM images. Near-field AFM-tip enhanced fluorescence lifetime imaging (AFM-FLIM) holds great promise for obtaining fluorescence images beyond the optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond the diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging living bacterial cells, we demonstrate a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging to characterize cell polarity.