Single-Molecule Characterization of Photophysical and Colloidal Properties of Biocompatible Quantum Dots

Colloidal semiconductor nanocrystals (NCs) have recently been introduced as novel fluorescent labels for various biological applications. Their unique optical properties — tunable narrow emission spectrum, broad excitation spectrum, high photostability and long fluorescent lifetimes (on the order of tens of nanoseconds) — make them attractive probes in experiments involving long observation times, multicolor and time-gated detection. Photophysical properties were investigated at the ensemble and single-molecule level for CdSe or CdTe core, CdSe/ZnS core-shell and surface-modified NCs. The use of NCs as fluorescent probes in biological applications requires various synthesis routes and surface modifications to enable solubility in aqueous solution and to allow labeling of biological macromolecules. Due to NC’s sensitivity to surface-defects chemical treatments have a significant influence on photophysical properties and need to be thoroughly monitored. Single-molecule fluorescence detection was used to characterize NC fluorescence, observe intermittency in the emission (blinking), and unravel a non-fluorescent subpopulation of NCs whose fluorescence can be restored through photo-induced activation. Stage-scanning confocal, epifluorescence and objective-type total-internal-reflection microscopy, were applied to observe surface-immobilized NCs. Fluorescence lifetimes were determined to be around 20 ns for single particles showing deviations from a mono-exponential decay. This observation was proven to be characteristic of single particles by monitoring photon antibunching. Fluorescence correlation spectroscopy (FCS) was used to characterize photophysical and colloidal properties in solution. It was shown to be a powerful technique to rapidly evaluate information on synthesis and surface modifications. These are essential to achieve water-solubility and bio-conjugation and dramatically influence the optical performance. FCS allows measuring concentration of fluorescent particles, an average brightness and particle size. From these observables, the brightness per particle can be estimated, something not possible in ensemble measurements due to the presence of absorbing but dark particles. The ratio of dark to fluorescent NCs was estimated and concentration changes due to photo-induced activation were observed. Particle sizes measured by FCS were compared to transmission electron microscopy and found in good agreement down to 7 nm. The correlation amplitude was observed to be excitation power dependent which was attributed to saturation and optical trapping effects. An electronic polarizability was evaluated and found to be two orders of magnitude larger than reported for measurements at non-resonant wavelength. Monte-Carlo simulations (MCS) were performed to compute autocorrelation functions under the influence of power-law blinking. Diffusion through a confocal volume, excitation-emission cycles with defined rate constants and on/off blinking were incorporated into MCS and used to investigate influences of saturation, size-distributions, photobleaching and blinking. The results were compared to experimental data of various NCs. Simulations account for both types of experimentally observed effects of blinking in FCS curves: significant deviation from a diffusion-model observed at high excitation powers; and no deviation from a diffusion-model despite the existence of blinking, observed at low excitation powers. Simulations showed that blinking does not influence FCS data for certain power law parameters