Near-field optical microscopy for DNA studies at the single molecular level

An aperture-type near-field optical microscope (NSOM) with two polarization detection channels has been used to image fluorescently labelled DNA with high spatial resolution and single molecule fluorescence sensitivity. The sample has been engineered such that there is o­nly o­ne rhodamine dye per DNA strand. Lateral and vertical DNA dimensions in the shear-force image are 14 +/- 2 nm and 1.4 +/- 0.2 nm, respectively. No sample deformation was observed under our imaging conditions. Near-field fluorescence imaging of individual fluorophores shows an optical resolution of 70 nm at full-width at half- maximum. Large intensity differences between individual rhodamine molecules attached to DNA are observed from the NSOM images. Statistics o­n rhodamine dyes in different environments (attached to glass, embedded in a polymer layer and attached to DNA) show bleaching rates of 10(-5). Total intensity line profiles together with in-plane angle orientation are used to characterize individual dyes. Rhodamine dyes show strong intensity fluctuations independent of the particular environment. These results are in contrast with the more stable photophysical behaviour as observed for carbocyanine molecules embedded in polymer matrices. The mobility of rhodamine-both lateral and rotational-is clearly influenced by its immediate surrounding and attachment to the surface.