Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells.
We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient 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 signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.
Micic
Miodrag
M
Fundamental Science Division, Pacific Northwest National Laboratory, MSIN K8-88, P.O. Box 999, Richland, WA 99352, USA.
Hu
Dehong
D
Suh
Yung Doug
YD
Newton
Greg
G
Romine
Margaret
M
Lu
H Peter
HP
D001419:Bacteria / Q000166:cytology*; D018407:Bacterial Physiological Phenomena; D016764:Cell Polarity; D018625:Microscopy, Atomic Force / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D020592:Shewanella / Q000502:physiology; D013499:Surface Properties