Abstract
Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.
Highlights
Single-molecule localization microscopy (SMLM) has become a popular technique to investigate the molecular structures, spatial distribution, clustering, and diffusion of receptors below the diffraction limit of light, allowing the observation of many biological phenomena never seen prior to the realization of these breakthrough techniques[1,2,3,4]
Specific and monovalent quantum dots enable fluorescence imaging in cells via DNA hybridization To produce specific and monovalent QDs by steric exclusion[27], CdSe:ZnS QDs that emit at 585 nm (QD585) were phase transferred from the organic to aqueous phase and wrapped with a polymer, a 50adenosine phosphorothioate DNA domain with a 20-nucleotide linker strand and a 20nucleotide imager strand (Fig. 1a)
We chose EGFR as a target molecule to apply QD-PAINT and test its applicability in cell imaging with improved spatial resolution compared with the current SMLM resolution
Summary
Single-molecule localization microscopy (SMLM) has become a popular technique to investigate the molecular structures, spatial distribution, clustering, and diffusion of receptors below the diffraction limit of light, allowing the observation of many biological phenomena never seen prior to the realization of these breakthrough techniques[1,2,3,4]. The current SMLM techniques frequently utilize photoswitchable organic dyes that allow achievable resolutions of 20–40 nm at best[12]. QDs with 10–20 times higher fluorescence intensity than organic dyes can achieve an ~3–5-fold improvement in resolution. Despite these excellent photophysical properties, an SMLM method that can distinguish many different molecules within the diffraction-limited region while taking advantage of the full potential of the substantially high photon yield of QDs to improve the resolution beyond the current limit of SMLM has not been reported[22,23,24]. If QDs with a substantially high photon output could be properly utilized for SMLM by conferring photoswitchability, specificity, and monovalency on them, the current resolution limit in photoswitchable organic dye-utilizing SMLM would be overcome
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