This study presents a molecular ruler based on plasmon coupling of single gold and silver nanoparticles for monitoring distances between single pairs of nanoparticles. The plasmon ruler allows continuous monitoring of separations up to 70 nm for over 3000 seconds. The optical properties of colloidal gold and silver nanoparticles are used to detect conformational changes and intramolecular distances of single biomolecules. The plasmon resonance wavelength of a metal nanoparticle is affected by other nanoparticles in its immediate environment. When two nanoparticles are brought into proximity, their plasmons couple, which shifts the resonance wavelength depending on the particle separation. This effect is used to monitor the directed assembly of gold and silver nanoparticle dimers in real time and to study the time dynamics of single DNA hybridization events. The plasmon ruler is used to follow the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level. DNA was chosen as the model system for four reasons: it has been used successfully to assemble discrete nanostructures, plasmon coupling has been demonstrated in bulk experiments to detect specific DNA targets, the length of DNA can be readily chosen, controlled, and is well understood, and the kinetics of single DNA hybridization is relevant for biotechnology and nanoscience applications. The plasmon ruler has several key advantages over FRET based rulers, which should allow a wide range of new in vitro single molecule experiments. The plasmon ruler neither blinks nor bleaches and does not depend on the relative probe orientation. Moreover, metal nanoparticles are good labels for electron and x-ray microscopy, which permits the development of novel multimodal imaging techniques. In general, gold and silver particles are more stable under physiological conditions than organic dyes. The plasmon resonance, however, does also depend on the refractive index of the surrounding, and it can be difficult to distinguish resulting redshifts from distance changes, so refractive index needs to be carefully controlled. The plasmon ruler has the potential to become an alternative to FRET for in vitro single molecule experiments, especially for applications demanding long observation times or large distances. In addition, analytical bulk assays based on particle aggregation can now be extended to the single molecule level, enhancing their sensitivity and allowing parallel processing. The plasmon ruler is used to monitor the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level. The plasmon ruler is used to follow the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level. The plasmon ruler is used to follow the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level.This study presents a molecular ruler based on plasmon coupling of single gold and silver nanoparticles for monitoring distances between single pairs of nanoparticles. The plasmon ruler allows continuous monitoring of separations up to 70 nm for over 3000 seconds. The optical properties of colloidal gold and silver nanoparticles are used to detect conformational changes and intramolecular distances of single biomolecules. The plasmon resonance wavelength of a metal nanoparticle is affected by other nanoparticles in its immediate environment. When two nanoparticles are brought into proximity, their plasmons couple, which shifts the resonance wavelength depending on the particle separation. This effect is used to monitor the directed assembly of gold and silver nanoparticle dimers in real time and to study the time dynamics of single DNA hybridization events. The plasmon ruler is used to follow the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level. DNA was chosen as the model system for four reasons: it has been used successfully to assemble discrete nanostructures, plasmon coupling has been demonstrated in bulk experiments to detect specific DNA targets, the length of DNA can be readily chosen, controlled, and is well understood, and the kinetics of single DNA hybridization is relevant for biotechnology and nanoscience applications. The plasmon ruler has several key advantages over FRET based rulers, which should allow a wide range of new in vitro single molecule experiments. The plasmon ruler neither blinks nor bleaches and does not depend on the relative probe orientation. Moreover, metal nanoparticles are good labels for electron and x-ray microscopy, which permits the development of novel multimodal imaging techniques. In general, gold and silver particles are more stable under physiological conditions than organic dyes. The plasmon resonance, however, does also depend on the refractive index of the surrounding, and it can be difficult to distinguish resulting redshifts from distance changes, so refractive index needs to be carefully controlled. The plasmon ruler has the potential to become an alternative to FRET for in vitro single molecule experiments, especially for applications demanding long observation times or large distances. In addition, analytical bulk assays based on particle aggregation can now be extended to the single molecule level, enhancing their sensitivity and allowing parallel processing. The plasmon ruler is used to monitor the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level. The plasmon ruler is used to follow the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level. The plasmon ruler is used to follow the directed assembly of functionalized particle pairs and to study the dynamics of DNA hybridization on a single molecule level.