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DNA condensation was only observed after the addition of Hoechst 33258 (H33258) among various types of DNA binding molecules. The morphological structural change of DNA was found to depend on the H33258 concentration. On comparison of fluorescence spectrum measurements with AFM observation, it was found that fluorescence quenching of DNA-H33258 complexes occurred after DNA condensation. Additionally, we showed that DNA condensation by H33258 was independent of sequence selectivity or binding style using two types of polynucleotides, i.e. poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Moreover, it was concluded that the condensation was caused by a strong hydrophobic interaction, because the dissolution of condensed DNA into its native form on dimethyl sulfoxide (DMSO) treatment was observed. This study is the first report, which defines the DNA condensation mechanism of H33258, showing the correlation between the single molecule scale morphology seen on AFM observation and the bulky scale morphology observed on fluorescence spectroscopy.

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.

Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographical and optical images of human chromosomes using a sharp and bent optical fiber as a near-field optical probe. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. The SNOAM system does not need pretreatment of samples such as metal coating or chemical immobilization. Near-field topographic and fluorescence images provided useful information on native chromosome structure.

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