Comparative imaging of a bacterial surface-located GFP fusion protein by epifluorescence and scanning near-field optical microscopy.

IcsA is an autotransporter protein that plays a role in the virulence of Shigella bacteria. We have examined the cellular localization of a fusion of an IcsA fragment to the green fluorescent protein (GFP) expressed in Escherichia coli using a dual epifluorescence and scanning near-field optical microscope. By combining the data obtained from far-field with near-field microscopy of the same sample, discrimination between surface-bound fusion proteins and fusion proteins located in the cellular cytoplasm becomes possible. Furthermore, and for the first time, the inherent advantages in resolution of the near-field images provides highly specific details of the location of a GFP fusion protein on a bacterial cell surface.