Scanning Near-Field Optical/Atomic Force Microscopy for Fluorescence Imaging and Spectroscopy of Biomaterials in Air and Liquid: Observation of Recombinant Escherichia coli with Gene Coding to Green Fluorescent Protein*

Abstract

We have developed a system of scanning near-field optical/atomic force microscopy (SNOM/AFM) for fluorescence imaging and spectroscopy of biomaterials in air and liquid. SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Optical resolution of SNOM images shows about 50 nm in an illumination mode for a standard sample of a patterned chromium layer of 20 nm thickness on a quartz glass plate. The SNOM/AFM system contains a photon counting system and polychrometer/ICCD (intensified charge coupled device) system for observation of the fluorescence image and spectrograph of micro areas, respectively. The gene coding to green fluorescence protein (GFP) was cloned in recombinant Escherichia coli (E. coli). Topography, fluorescence image and spectrograph of recombinant E. coli by SNOM/AFM showed a difference in fluorescence in individual E. coli. Fluorescence activity of GFP can thus be used as a convenient indicator of transformation. SNOM/AFM is also applicable to observe immobilized E. coli on a glass plate in water with a liquid chamber and may allow the viewing of observation of floating organisms.