Maximum-likelihood position sensing and actively controlled electrokinetic transport for single-molecule trapping Lloyd Davis, Zbigniew Sikorski, William Robinson, Guoqing Shen, Xiaoxuan Li, Brian Canfield, Isaac Lescano, Bruce Bomar, William Hofmeister, James Germann, Jason King, Yelena White, and Alexander Terekhov Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN 37388 ABSTRACT 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. Keywords: Single-molecule spectroscopy, single-molecule trapping, maximum-likelihood, photon counting, pulse- interleaved excitation 1. INTRODUCTION Since the first report of the detection of individual single-chromophore molecules in solution [1], applications and advanced techniques for single-molecule detection (SMD) have rapidly developed, including the means for wide-field imaging of single molecules with sub-diffraction precision, for tracking fluorescently-labeled biomolecules in 2 dimensions (2-D) on cellular surfaces [2], and for observing motion between different focal planes [3]. For many applications, confocal microscopy is preferable to wide-field imaging as it provides improved signal-to-noise due to the very small detection volume; it is necessary for two-photon excitation, which offers the advantage of inherent sectioning and no excitation of out-of-plane molecules; and it facilitates monitoring of sub-nanosecond fluorescence lifetimes and sub-millisecond dynamics by use of single-photon avalanche diode (SPAD) detectors for time-resolved single-photon counting and fluorescence correlation spectroscopy. However, a freely diffusing molecule quickly leaves the confocal volume. Tracking and trapping can enable the molecule to be kept within the detection volume until it ultimately photobleaches. A means for 2-D tracking and trapping of a single fluorescent molecule within a confocal microscope by rotating the laser spot about the molecule to sense its position from the phase and amplitude of modulation of the fluorescence signal and then translating the center of rotation or the sample in response to the molecule’s diffusion was suggested some years ago [4] and several groups have since developed the capability for trapping in 2-D by this method [5]. The method has been extended to 3-D tracking of fluorescent particles by use of orbits in different z-sections to track axial motion [6- 9]. In most cases, the response time of the piezoelectric translation stage used to recenter the molecule limits the applicability of the trap to slowly-diffusing species, but the time required for the position sensing, which is determined by the speed of the rotating laser focus and ultimately the photon count rate, can also provide a limitation. The use of acousto-optic beam deflectors to rotate the laser focus at ~50 kHz and electrophoretic and electro-osmotic forces to more quickly recenter the fluorescent particle has been shown to provide advantages for 2-D trapping [10]. For nanoscale objects, electrokinetic forces are stronger than magnetic, ac dielectrophoretic, or optical forces, and potentially enable a feedback system faster than one based on a translation stage. Single Molecule Spectroscopy and Imaging edited by Jörg Enderlein, Zygmunt K. Gryczynski, Rainer Erdmann Proc. of SPIE Vol. 6862, 68620P, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.763833 Proc. of SPIE Vol. 6862 68620P-1