Advancement of Blinking Suppressed Quantum Dots for Enhanced Single Molecule Imaging
Dr. Shuming Nie, Advisor - School of Biomedical Engineering, Georgia Institute of Technology
Dr. Younan Xia - School of Biomedical Engineering, Georgia Institute of Technology
Dr. Mostafa El-Sayed - School of Chemistry, Georgia Institute of Technology
Dr. Gang Bao - School of Biomedical Engineering, Georgia Institute of Technology
Dr. Adam Marcus - School of Oncology, Emory University
Semiconductor nanocrystalline quantum dots (QDs) have been intensely studied in bioimaging applications due to their exceptional optical properties such as size-tunable narrow emission spectra, broad absorption envelopes, and high resistance to chemical and photodegradation in comparison to conventional fluorophores or fluorescent proteins. An inherent problem that quantum dots have shared with other single emitters is fluorescent intermittency or blinking. The blinking of a single molecule or single QD refers to random transitions amid absorption and emission cycles followed by sustained intervals of time without fluorescence regardless of continuous laser excitation. Frequent and prolonged off times raise difficulties in correspondence between frames when tracking the position of single molecules in cellular experiments due to cells not being homogeneous vessels with a single diffusion coefficient. In the last six years there has been success suppressing the blinking of quantum dots in practice, but were limited to thick shell particles with large diameters, gradient dots with overlapping multi-peak emission, or immersing the probes in specific solutions of reducing reagents. All of these methods present major difficulties when considering application to live molecular tracking experiments. Within the past year, however, efforts have demonstrated success in overcoming a few of these hurdles, but still lack a general methodology in creating blinking suppressed probes. Among these reports, only one to date has demonstrated single molecule tracking potential.
This study composes a generalizable framework by means of physical-chemical theory undertaking the mechanisms of fluorescent intermittency and synthesis measures designed in light of this information, to develop blinking suppressed particles which are better suited for single molecular imaging than currently available QDs and validates their use in a biological setting. Such probes were found to provide superior frame correspondence in trajectory reconstruction of tracking studies due to having short infrequent off times. Controlling blinking towards brief and infrequent off-times offer negligible signal loss, leading to continuous dynamical information at higher acquisition rates. In addition to the enhanced on times, the emission spectra were observed to be single peaked with thin full width at half maximums (FWHMs) due to synthesis techniques preserving monodispersity, thus permitting multiplexed single molecule tracking of different species.
The innovation of the study is synthesizing QDs within a general framework that have greatly diminished fluorescent off-times while preserving small sizes, solution independency, and no multi-peak emission. The work presented yields the following outcomes: First, it establishes a general structure based on theoretical forethought in which to synthesize blinking suppressed imaging probes. Next, synthesis techniques are developed to produce QDs with the structure outlined within the general framework. Finally, a comparative test of blinking suppressed to conventional core/shell particles elucidating the benefits is shown in a single molecule tracking experiment. These studies present that when adhering to the prescribed framework outlined, one can produce blinking suppressed QDs with sensible sizes for single molecule tracking experiments with minimal signal loss.