(199c) Photoswitchable Quantum Dots Probes for Superresolution Microscopy

Superresolution microscopy has been developed as a tool to image native states of biological structures beyond the diffraction limit of light. Although electron microscopy techniques have surpassed this limit, they lack live imaging capability. Stochastic optical reconstruction microscopy (STORM) involves reconstruction of multiple images of localized fluorescent probes stochastically switching between light and dark states. Unlike many other superresolution imaging techniques, STORM permits discrete imaging at high densities. Unfortunately, most fluorescent probes designed for STORM provide limited fluorescence signals as a result of quenching, low brightness and contrast between dark and light states, poor stability, and short lifetimes. This research aims to develop a robust fluorescent probe to overcome prior limitations by using photoswitchable quantum dots (QDs).

QD probes were designed using the principles of Förster resonance energy transfer (FRET) by radiative transfer of the QD (donor) fluorescence to a light sensitive material (acceptor) to achieve a dark state. Previous studies have shown that gold nanoparticles (AuNPs) are excellent acceptors, especially for QDs. Additionally, FRET is strongly dependent on the distance between the donor and acceptor. Therefore, the probe consisted of QD-AuNP pairs formed using a DNA linker, whose length can be precisely controlled by the number of base pairs. The QD-AuNP pairs were therefore synthesized by conjugating the nanoparticle surfaces with complementary single stranded-deoxyribonucleic acid (ss-DNA). Reversible photoswitching was induced in these probes by utilizing photo-sensitive azobenzene modified DNA strands that can induce reversible DNA hybridization through cis-trans conversions induced by light. In this research, QD coatings were optimized to reduce the QD-AuNP separation distance and QD-AuNP spectral overlap were optimized. Both are crucial for efficient FRET. These probes are being analyzed using fluorescence spectroscopy by simultaneously modulating the photo-sensitive-DNA with a laser source to measure quenching efficiency. The optimized probes developed from this study will then be tested by comparative STORM imaging.