3D single particle tracking spectroscopy is a technique which allows a person to follow a freely diffusing nanometer-sized particle in three dimensions, in real-time. The basic scheme involves illuminating a nanometer-sized probe with a confocal microscope, optically detecting its position change with avalanche photodiodes, and then moving a piezo-electric stage to counteract the particle's movement. The tracked particle is centered within the collection volume of a confocal microscope, enabling concurrent spectroscopic readouts of the tracked particle's emission.

The motivation for this technique is the desire to perform spectroscopy in complex environments. Dynamics in complex environments, such as within a living cell, occur over a range of time and length scales. Unlike homogeneous systems, the multiple dynamical length and time scales are correlated, prohibiting the use of simplifications like timescale separation. In order to understand the correlations across different scales, new experimental techniques are needed which can provide long-time observation and spatially-correlated dynamical measurements. 3D single particle tracking spectroscopy is the realization of this dream for nano-scale environments. Counteracting the particle's diffusion with the piezo stage enables long-time observation of the freely diffusing particle. Because the current stage position is a measure of the particle's position, one can perform spatially-correlated spectroscopy.

The scientific impact of this thesis has been to develop the 3D single particle tracking spectroscopy technique, demonstrate the potential of using nanoparticles to design new kinds of single-molecule/probe assays, and open the possibility of using an under-appreciated class of bright, photo-stable nanoparticles (large gold nanoparticles) for biophysical applications.