Three-dimensional position determination of nanoparticles using a two-photon microscope

We are developing a means to extend the two-photon microscope to enable three-dimensional sub-diffraction measurement of the position and trajectory of a single nanoparticle as it traverses the probe volume. By use of a Ti-Sapphire laser and a double-Mach-Zehnder interferometer configuration, four laser beams with temporally interleaved pulses are created. These are tightly focused by a 1.2-NA water-immersion microscope objective to four overlapping volumes centered at slightly offset points arranged in a tetrahedron. Fluorescence from the four-focus probe volume is then collected onto a single-photon avalanche diode and the photon time stamps are recorded. Time-resolved photon detection with maximum-likelihood analysis is thereby used to determine the position of the nanoparticle from the relative intensities of fluorescence from each of the four foci.~ We present measurements of the profile of the four-focus configuration and results from calibration experiments obtained by translating a single gold nanodot or a fluorescent nanobead through the probe volume. Application of the position determination to single-particle trapping is also briefly discussed.