Tracking Real-Time Nanoparticle Positions and Measuring Three-Dimensional Solution Flow with a Four-Focus Confocal Microscope

This dissertation presents the development of instrumentation for measuring the position of a single emitter within the sample volume of a confocal fluorescence microscope with sub-diffraction limited precision in three dimensions together with applications for determining solution flow and for tracking a fluorescent nanoparticle as it undergoes Brownian diffusion. The localization method is based on comparing photon counts from alternating excitation of the emitter by four laser beams, which are focused at slightly offset positions in a tetrahedral pattern within the confocal volume. Two experimental set-ups are constructed. In the first, the four beams are from a femtosecond laser, which provides two-photon excitation (2PE) of a nanomolar solution of rhodamine B. Time-resolved photon counting into four channels and cross-correlation of the channels yields a set of sixteen curves that vary with the flow of solution through the tetrahedral pattern. Standard fluorescence correlation spectroscopy fitting methods are extended to model solution flow and a Gaussian-Lorentzian beam profile for 2PE. Global fitting of the sixteen curves to extract the flow is studied using data from a collaborator’s computer simulation. The model successfully fits simulated data with flow in one dimension, but accuracy is found to be poor when simultaneously fitting three velocity components. In the second set-up, the four beams are provided by 635-nm fiber-coupled laser diodes. LabVIEW real-time software alternately pulses the diodes, performs time-gated photon counting to estimate the nanoparticle position, and controls a piezo stage to track its motion as it undergoes diffusion. The confocal microscope enables tracking of nanoparticles with diffusivity exceeding 10 µm² [micron²]s^-1, currently limited by the 1.8 ms update interval of the piezo stage, while also providing exceptional sensitivity, with a net fluorescence detection efficiency of about 6%, comparable to that of a highly optimized single-molecule microscope.