Analysis of individual (macro)molecules and proteins using near-field optics

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Abstract
Recent achievements in single molecule detection using near-field optical excitation are presented. By proper control of technology, distinct advantages of near-field optics are exploited: (i) the nanometric excitation/emission volume (104–105 nm3), which provides high spatial resolution, localization of a single molecule within a few nm, and reduced background; (ii) the sensitivity for single molecule orientation in all three dimensions; (iii) the high local brightness, allowing real-time single molecule detection down to μs resolution; (iv) the simultaneous colocalization with nanometric surface topography. Real-time quantum jumps between singlet and triplet state of an individual molecule are observed. Distributions for triplet state lifetime and crossing yield are determined. Both triplet state lifetime and crossing yield of a single molecule appear to vary in time, due to the local heterogeneity. Individual dendritic molecules containing a single fluorescent core are investigated. The dendritic assemblies are discriminated from free fluorescent cores on the basis of accurate simultaneous localization of both the fluorescent core and the topography of the surrounding dendritic shell. Intramolecular rotational motion of the fluorescent core is observed. Individual green fluorescent proteins are visualized, both in fluorescence and topography. Photoinduced conformational changes to a nonemissive form of the protein are observed, leading to long dark intervals of several seconds.