人工膜と生体膜の接点を探る 最近の膜研究の方法 原子間力制御型近視野顕微鏡による生体イメージング

Abstract

A scanning near-field optical/atomic-force microscope (SNOAM) system was applied to 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 (AFM) cantilever and a scanning near-field optical microscopy (NSOM) probe. Optical resolution of this system indicated about 50-100 nm in a fluorescence mode for fluorescent latex beads on a quartz glass plate. The SNO-AM system used 458 or 488 nm from Ar ion laser multiline of excitation of green fluorescent protein (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 fluoresence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. 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, individual E. coli cells expressed different fluorescent intensity. Fluorescence obtained by SNOAM indicated GFP oxidation possibly occured near cell surface. SNOAM also provided us with simultaneous topographical and optical images of human chromosomes. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. Topographic images clearly indicated duplicated structure on metaphase chromosome, while fluorescence images were a different shape probably because it depended on the combination of SYBRTM Green I and chromosome DNA. Atomic force images have some artefacts, however they can be corrected by comparison with the fluorescence image. Topography and fluorescence images of RBL-2H3 mast cells surface were determined with/without DNP-BSA stimulation by SNOAM system. Near-field fluorescence images were obtained from the granules stained with quinacrine. Fluorescence profiles and intensities were largely changed after allergen stimulation. Exocytotic events of granules were specially discussed based on SNOAM.