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Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and the centers of the two probes are mapped onto the same space. Multiple measurements can be made before the fluorophores photobleach, allowing intramolecular and intermolecular distances to be tracked through time. This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy. The instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances. A dual-labeled duplex DNA molecule (30 bp) was used as a 10-nm molecular ruler to confirm the validity of the method. We also used SHREC to study the motion of myosin V. We directly observed myosin V's alternating heads while it walked hand-over-hand along an actin filament.centroid tracking ͉ dynamic conformational changes ͉ fluorescence ͉ molecular motors ͉ total internal reflection microscopy S ingle molecule fluorescence techniques are bridging the areas of structural biology and biochemistry (1). They yield structural constraints for an enzyme during its biochemical cycle. To probe close to this interface between structure and function, distances must be resolved on the order of the size of the enzyme. However, far field fluorescence microscopy is limited in its resolution by the Rayleigh criterion at Ϸ250 nm. On the other end of the size spectrum, single molecule FRET (smFRET) provides a way to estimate intramolecular distances Ͻ10 nm and has yielded insights on a range of biological molecules from the Tetrahymena ribozyme to the ribosome (2-4).Recent methods, single-molecule high-resolution imaging with photobleaching (SHRImP) and nanometer-localized multiple single-molecule (NALMS), have been developed in an attempt to bridge this ''gap'' in distance resolution. These methods are capable of measuring an intramolecular distance to high precision, but only once, because of the intrinsic destruction of one of the probes (5, 6). A time series of the intramolecular distance is not possible with these methods, removing one of the greatest potential benefits of single molecule imaging.Addressing the gap in imaging techniques suitable for studying macromolecules, we introduce a technique, single-molecule high-resolution colocalization (SHREC) of fluorescent probes, that can precisely measure intramolecular and intermolecular distances through time. By separately imaging two chromatically different fluoropho...

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