Scanning near-field optical/atomic-force microscopy for biomedical applications

We have developed Scanning Near-field Optical / Atomic force Microscopy(SNOM/AFM). The SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Resonant frequency of the optical fibercantilever is 15-40 kHz. Optical resolution of the SNOM/AFM images shows less than 50 nm. The SNOM/AFM system contains photon counting system and polychrometer/ICCD system to observe fluorescence image and spectrograph of micro areas, respectively. A SNOM-AFM system was newly applied to analyses of biological samples. In this system a feedback signal from AFM in the noncontact mode was used to scan the probe tip along the surface contour of the sample. An optical fiber with a sharp tip on one end was bent for use as cantilever, and ac amplitude of the cantilever deflection was held constant during scanning by moving the stage. Green Fluorescent Protein(GFP) absorbs blue light and emits green light. GFP should be a convenient indicator of transformation and one that could allow cells to be separated with fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant E. coli and plant cells. Spatial distribution of GFP gene expression was clarified using a SNOM-AFM system. Fluorescent spectroscopic analyses supported GFP was surely produced in E. coli and plant cells. Applications to gene identification in human genomes were also discussed.