James A. Germann

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.

Three-dimensional tracking of a single fluorescent nanoparticle using four-focus excitation in a confocal microscope.

We report high sensitivity detection and tracking of a single fluorescent nanoparticle in solution by use of four alternately pulsed laser diodes for fluorescence excitation in a confocal microscope. Slight offsets between the centers of the overlapping laser foci together with time-resolved photon counting enable sub-micron precision position measurements. Real-time correction for diffusional motion with a xyz-piezo stage then enables tracking of a nanoparticle with diffusivity up to ~12 μm(2) s(-1). Fluorescence correlation spectroscopy and calibration measurements indicate a net fluorescence photon detection efficiency of ~6-9%, comparable to that of an optimized single-molecule microscope.

Four-focus single-particle position determination in a confocal microscope

We discuss the capabilities for sub-diffraction, single-nanoparticle position determination in a confocal one- or twophoton microscope with four-focus pulse-interleaved excitation and time-gated single-photon counting. As the technique is scalable to multiple detectors for multi-color observations, it can be used to find the separations of differently colored molecules over a distance range that is complementary to that achievable by FRET. Also, there is a possibility for improved spatial localization by using the nonlinearity of saturation of the excitation or by using the technique together with imaging of the point spread function. Applications of two experimental set-ups for four-focus fluorescence excitation for studies of quantum dots and single-particle manipulation and trapping are also discussed.