Maximum-likelihood position sensing and actively controlled electrokinetic transport for single-molecule trapping

A freely diffusing single fluorescent molecule may be scrutinized for an extended duration within a confocal microscope by actively trapping it within the femtoliter probe region. We present results from computational models and ongoing experiments that research the use of multi-focal pulse-interleaved excitation with time-gated single photon counting and maximum-likelihood estimation of the position for active control of the electrophoretic and/or electro-osmotic motion that re-centers the molecule and compensates for diffusion. The molecule is held within a region with approximately constant irradiance until it photobleaches and/or is replaced by the next molecule. The same photons used for determining the position within the trap are also available for performing spectroscopic measurements, for applications such as the study of conformational changes of single proteins. Generalization of the trap to multi-wavelength excitation and to spectrally-resolved emission is being developed. Also, the effectiveness of the maximum-likelihood position estimates and semi-empirical algorithms for trap control is discussed.