NEAR-FIELD OPTICAL MICROSCOPY FOR DNA STUDIES AT THE SINGLE MOLECULAR-LEVEL

An aperture-type near-field optical microscope (NSOM) with two polariz ation detection channels has been used to image fluorescently labelled DNA with high spatial resolution and single molecule fluorescence sen sitivity. The sample has been engineered such that there is only one r hodamine dye per DNA strand. Lateral and vertical DNA dimensions in th e 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 di fferences between individual rhodamine molecules attached to DNA are o bserved from the NSOM images. Statistics on rhodamine dyes in differen t environments (attached to glass, embedded in a polymer layer and att ached to DNA) show bleaching rates of 10(-5). Total intensity line pro files together with in-plane angle orientation are used to characteriz e individual dyes. Rhodamine dyes show strong intensity fluctuations i ndependent of the particular environment. These results are in contras t with the more stable photophysical behaviour as observed for carbocy anine molecules embedded in polymer matrices. The mobility of rhodamin e-both lateral and rotational-is clearly influenced by its immediate s urrounding and attachment to the surface.