Improved resolution in single-molecule localization microscopy using QD-PAINT
Open Access
- 2 March 2021
- journal article
- research article
- Published by Springer Science and Business Media LLC in Experimental & Molecular Medicine
- Vol. 53 (3), 384-392
- https://doi.org/10.1038/s12276-021-00572-4
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.Keywords
Funding Information
- National Research Foundation of Korea (NRF-2016K1A1A2912722, NRF-2019R1A2C2002152)
This publication has 45 references indexed in Scilit:
- Fluorescence nanoscopy in cell biologyNature Reviews Molecular Cell Biology, 2017
- The 2015 super-resolution microscopy roadmapJournal of Physics D: Applied Physics, 2015
- Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINTNature Methods, 2014
- Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in AxonsScience, 2013
- Maturation-Dependent HIV-1 Surface Protein Redistribution Revealed by Fluorescence NanoscopyScience, 2012
- Extending Microscopic Resolution with Single-Molecule Imaging and Active ControlAnnual Review of Biophysics, 2012
- Nanoscale architecture of integrin-based cell adhesionsNature, 2010
- Probing cellular events, one quantum dot at a timeNature Methods, 2010
- Imaging Intracellular Fluorescent Proteins at Nanometer ResolutionScience, 2006
- Antibody Domain Exchange Is an Immunological Solution to Carbohydrate Cluster RecognitionScience, 2003