Optically monitoring the mechanical assembly of single molecules

Bottom-up assembly at the level of individual molecules requires a combination of utmost spatial precision and efficient monitoring. We have previously shown how to 'cut-and-paste' single molecules, and other groups have demonstrated that it is possible to beat the diffraction limit in optical microscopy. Here we show that a combination of single-molecule cut-and-paste surface assembly, total internal reflection fluorescence microscopy and atomic force microscopy can be used to deposit individual fluorophores in well-defined nanoscale patterns and also to monitor the process in real time with nanometre precision. Although the size of the pattern is well below the optical resolution of the microscope, the individual dyes are identified by localizing the centroids and detecting the photobleaching of the fluorophores. With this combination of methods, individual dyes or labelled biomolecules can be arranged at will for specific functions, such as coupled fluorophore systems or tailored enzyme cascades, and monitored with nanoscale precision.