High‐Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super‐Resolution Imaging

Abstract

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4-9 frame binning, we achieved a 2-3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

Hyperbranched Polymer Dots with Strong Absorption and High Fluorescence Quantum Yield for In Vivo NIR-II Imaging

“…Driven by the clinical needs, a wide range of in vivo probes have been explored, including nanoparticles, − small-molecule dyes, − etc. Among these materials, semiconducting polymer dots (Pdots) have been demonstrated as a class of promising fluorescent probes due to their excellent photostability, high single-particle brightness, good biocompatibility, etc. − Though Pdots with NIR-II emission have been reported, − their optical properties, including the absorption cross-section and the fluorescence quantum yield (QY), still require further optimization. Donor–acceptor–donor (D-A-D) fluorophores are promising NIR-II emitters, − but they typically become less emissive due to aggregation-induced quenching (ACQ) upon polymerization and formation of nanoparticles.…”