(196j) Synthesis of Photoswitchable Quantum Dots for Superresolution Microscopy

Superresolution microscopy is an excellent tool for imaging biological structures because it allows live imaging below the diffraction limit of light. Particularly, stochastic optical reconstruction microscopy (STORM) enables sub-diffraction limit imaging by reconstructing multiple images of localized fluorescence signals that stochastically switch between on and off states. However, most fluorescence probes used for STORM are limited by a short lifetime or quenching, limiting the number of signal containing frames that can be collected. In addition, the relatively low contrast between light and dark states of probes can affect the image quality. Quantum Dots (QDs) are semiconducting nanocrystals that exhibit a size-dependent fluorescence properties with a broad excitation and narrow emission. Studies have applied QDs for supreresolution microscopy, such as using QD photobleaching or blinking to control on/off signals. We previously demonstrated photoswitchable QDs for STORM by engineering QD and gold nanoparticle (AuNP) complexes to enable user controlled Förster resonance energy transfer (FRET), which is the radiative transfer of photons from the QD (donor) to adjacent AuNPs (acceptor), resulting in a dark state. To stochastically induce on/off states in QD-AuNP complexes, we conjugated both nanoparticles to complementary single-stranded deoxyribonucleic acid (ssDNA) strands. ssDNA was modified with a photo-responsive azobenzene group, which allowed reversible hybridization through cis-trans conformational changes induced by light. However, this method was not very reproducible and was inconsistent in the degree of quenching. Ideally, a FRET-based reporter probe should demonstrate > 95% quenching for optimal STORM imaging. In this work, we demonstrate two photoswitchable QD probe designs. In the first, instead of conjugating ssDNA to NPs via conventional conjugation techniques (i.e. EDC/Sulfo-NHS), we embedded ssDNA within the QD crystal shell, avoiding the need for a separate linker to serve as the conjugatation tag. In the second approach, we employed the “loops-trains-trails” technique, which offers more effective and compact surface coating minimizing the separation distance between neighboring QDs and AuNPs. ssDNA is then conjugated to this coating using traditional chemistries. Here, we report the results of our synthesis techniques, including QD stability after ssDNA functionalization, and the sensitivity and modulation efficiency of photo-responsive DNA suitable for STORM imaging.