Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules

Individual fluorescent polystyrene nanospheres (< 10-100-nm diameter) and i ndividual fluorescently: labeled DNA molecules were dispersed on mica and a nalyzed using time-resolved fluorescence spectroscopy and atomic force micr oscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (I) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microsco pe, (2) recording the time-resolved fluorescence emission, and (3) measurin g the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resul ted in concurrent high-resolution (similar to 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images ena bled unambiguous identification of the sample topography residing within th e excitation region of the optical microscope, facilitating the identificat ion and structural characterization of the nanoparticle(s) or biomolecule(s ) responsible for the fluorescence signal observed in step 2. These measure ments also provided the lateral position of the particles relative to the l aser excitation profile and the surrounding topography with nanometer-scale precision and the relationship bem een the spectroscopic and structural pr operties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.