After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theoretical background including particle identification, localization, and trajectory reconstruction. General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed. We then highlight some applications of SPT to study various biological and synthetic materials systems. Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT.

A strong intrinsic signal is advantageous over labeling for optical detection of nanoparticles. Intense scattering and absorption by the surface plasmon resonance, which exceeds molecular cross sections, provides a direct method for visualizing noble metal nanoparticles. While two-photon luminescence in gold nanoparticles yields a strong signal, one-photon luminescence is generally regarded to be much weaker and has seldom been employed for optical nanoparticle detection. In this article we investigated one-photon luminescence of gold nanospheres and nanorods using single particle spectroscopy with excitation at 514 and 633 nm. We characterized the polarization dependence, determined the quantum yield, and present a mechanism describing one-photon luminescence. Our results suggest fast interconversion between surface plasmons and hot electron–hole pairs and show that the luminescence occurs via emission by a surface plasmon. Using the information obtained from the single particle studies, we were able to successfully employ one-photon luminescence for correlation spectroscopy measurements and to correctly interpret auto- and cross-correlation functions, which were used to determine the hydrodynamic sizes of several gold nanoparticle samples and to extract rotational dynamics of nanorods. Because of the difference in size dependence for one-photon luminescence compared to scattering, luminescence correlation spectroscopy of metal nanoparticles is advantageous as it is not as strongly affected by the presence of larger nanoparticles or aggregates. This was verified by measuring luminescence as well as scattering correlation traces for a mixture of nanoparticles containing 98% 57 nm and 2% 96 nm gold nanospheres.

Plasmon-coupled circular dichroism has emerged as a promising approach for ultrasensitive detection of biomolecular conformations through coupling between molecular chirality and surface plasmons. Chiral nanoparticle assemblies without chiral molecules present also have large optical activities. We apply single-particle circular differential scattering spectroscopy coupled with electron imaging and simulations to identify both structural chirality of plasmonic aggregates and plasmon-coupled circular dichroism induced by chiral proteins. We establish that both chiral aggregates and just a few proteins in interparticle gaps of achiral assemblies are responsible for the ensemble signal, but single nanoparticles do not contribute. We furthermore find that the protein plays two roles: It transfers chirality to both chiral and achiral plasmonic substrates, and it is also responsible for the chiral three-dimensional assembly of nanorods. Understanding these underlying factors paves the way toward sensing the chirality of single biomolecules.

Addition of butylamine to a solution of colloidal CdSe nanoparticles (NPs) caused a decrease in fluorescence intensity, with no effect on the picosecond bleach recovery of the exciton formation or on the luminescence dynamics. The relative fluorescence quantum yield was found to decrease with increasing butylamine concentration and to level off at high concentrations, but the fluorescence lifetimes were not influenced. The nonlinear concentration dependence of the fluorescence quantum yield did not follow the Stern−Volmer equation. This is in agreement with the observation that the CdSe luminescence lifetime was not affected by the addition of butylamine. A mechanism is proposed in which the emission observed in CdSe is assumed to result from the combination of surface-trapped electrons and holes. n-Butylamine occupies hole sites, thus blocking the recombination process, which results in decreasing the density of luminescent centers. These results will be discussed in terms of the nature of the binding sites of the amine on the nanoparticle surface.

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.