John F. Lesoine, P.A. Venkataraman, Peter C. Maloney, Mark Dumont, Lukas Novotny


Fluorescent analysis of single molecules is commonly performed either by immobilizing

the molecule on a surface or by allowing the molecule to diffuse freely through a stationary laser focus. Surface immobilization is prone to perturbations of molecular behavior and the accuracy of measurement performed using single molecule diffusion is limited by the low number of photons emitted during passage through the illuminated volume. We present a method for single molecule recycling that circumvents these problems and allows the same molecule to be measured hundreds of times without surface tethering. The approach is based on the use of electroosmosis to drive transit of a single target molecule through a submicron fabricated channel in the focus of a fluorescence microscope. Following initial detection of a fluorescent molecule in the channel, reversal of an electrical potential following a controllable delay is used to repeatedly send the same molecule back through the microscope focus. For a given distribution of dye on-times before photo-blinking occurs, increasing the times between observations as we can do is the only way to extend the total observation time before a molecule is lost from the trap. The variation in the turnaround times may be used to measure the diffusion coefficient of one molecule. Other advantages of this single molecule recycling scheme are illustrated by demonstration of the ability to distinguish individual double-stranded DNAs of different lengths in a mixture DNAs of different lengths.