Simultaneous topographic and fluorescence imagings of recombinant bacterial cells containing a green fluorescent protein gene detected by a scanning near-field optical/atomic force microscope.

Abstract

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.