Three Dimensional Localization with Time-Gated Photon Counting and Maximum Likelihood Analysis

The localization of particles is an important step for the study of nanoscale objects and systems. Research is ongoing on techniques to find the position of particles moving about freely or interacting with other objects, especially below the diffraction limit of optical microscopy. The method of particle localization under development here arranges four laser beam foci created from a femtosecond pulsed Ti-Sapphire laser into a tetrahedron. The microscope constructed for this thesis includes optics to split each Ti-Sapphire laser pulse into four temporally and spatially separated pulses at the focus of the objective. Maximum likelihood analysis of the time-gated fluorescence photons then provides sub-diffraction localization of single emitters. Samples of Rhodamine B, fluorescent latex beads, and nanospheres of gold are evaluated to see if they provide adequate signal-to-noise with time-gated detection for localization measurements. For these measurements, samples in aqueous solution are allowed to freely diffuse through the focal volume. Fluorescence cross correlation spectroscopy measurements indicate that the four-foci microscope is sensitive enough to detect single emitters and can also be used to measure velocity. Also, samples are dried onto microscope coverslips and translated through the focal volume of the tetrahedron using a piezoelectric stage, so that the trajectory of a single emitter is controlled. Raster scanning measurements confirm that the time-gated photon counting hardware and Labview software can separate fluorescence photons into four channels corresponding to the four excitation foci. The instrument constructed for this thesis is to be used in experiments to track and trap a single fluorescent emitter in solution.