Improved resolution in single-molecule localization microscopy using QD-PAINT

다국어 초록 (Multilingual 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.

참고문헌 (Reference)

1 Strauss, S., "Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT" 17 : 789-791, 2020

2 Huang, F., "Ultra-high resolution 3D imaging of whole cells" 166 : 1028-1040, 2016

3 Hell, S. W., "The 2015 super-resolution microscopy roadmap" 48 : 443001-, 2015

4 Schickinger, M., "Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices" 115 : E7512-e7521, 2018

5 Uyeda, H. T., "Synthesis of compact multidentate ligands to prepare stable hydrophilic quantum dot fluorophores" 127 : 3870-3878, 2005

6 Li, H., "Switchable fluorophores for single-molecule localization microscopy" 118 : 9412-9454, 2018

7 Groc, L., "Surface trafficking of neurotransmitter receptor : comparison between single-molecule/quantum dot strategies" 27 : 12433-12437, 2007

8 Schnitzbauer, J., "Super-resolution microscopy with DNA-PAINT" 12 : 1198-, 2017

9 Jungmann, R., "Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami" 10 : 4756-4761, 2010

10 Gao, X., "Quantum-dot nanocrystals for ultrasensitive biological labeling and multicolor optical encoding" 7 : 532-537, 2002

11 Michalet, X., "Quantum dots for live cells, in vivo imaging, and diagnostics" 307 : 538-544, 2005

12 Byers, R. J., "Quantum dots brighten biological imaging" 45 : 201-237, 2011

13 Chan, W. C., "Quantum dot bioconjugates for ultrasensitive nonisotopic detection" 281 : 2016-2018, 1998

14 Xing, Y., "Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging" 4 : 307-319, 2008

15 Daeha Seo, "Production and Targeting of Monovalent Quantum Dots" MyJove Corporation (92) : 2014

16 Pinaud, F., "Probing cellular events, one quantum dot at a time" 7 : 275-285, 2010

17 Thompson, R. E., "Precise nanometer localization analysis for individual fluorescent probes" 82 : 2775-2783, 2002

18 Zhao, Q., "Photon budget analysis for fluorescence lifetime imaging microscopy" 16 : 086007-, 2011

19 Dai, M., "Optical imaging of individual biomolecules in densely packed clusters" 11 : 798-807, 2016

20 Kanchanawong, P., "Nanoscale architecture of integrin-based cell adhesions" 468 : 580-584, 2010

21 Werbin, J. L., "Multiplexed Exchange-PAINT imaging reveals liganddependent EGFR and Met interactions in the plasma membrane" 7 : 12150-, 2017

22 Jungmann, R., "Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT" 11 : 313-318, 2014

23 Xu, J., "Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy" 9 : 2917-2925, 2015

24 Howarth, M., "Monovalent, reduced-size quantum dots for imaging receptors on living cells" 5 : 397-399, 2008

25 Gao, J., "Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging" 7 : 2511-2519, 2015

26 Chojnacki, J., "Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy" 338 : 524-528, 2012

27 Jung, S., "Light-induced fluorescence modulation of quantum dot-crystal violet conjugates : stochastic off–on–off cycles for multicolor patterning and super-resolution" 139 : 7603-7615, 2017

28 Wäldchen, S., "Light-induced cell damage in live-cell super-resolution microscopy" 5 : 15348-, 2015

29 Saxton, M. J., "Lateral diffusion in an archipelago. Single-particle diffusion" 64 : 1766-1780, 1993

30 Gao, X., "In vivo cancer targeting and imaging with semiconductor quantum dots" 22 : 969-976, 2004

31 Betzig, E., "Imaging intracellular fluorescent proteins at nanometer resolution" 313 : 1642-1645, 2006

32 Farlow, J., "Formation of targeted monovalent quantum dots by steric exclusion" 10 : 1203-1205, 2013

33 Sahl, S. J., "Fluorescence nanoscopy in cell biology" 18 : 685-, 2017

34 Auer, A., "Fast, background-free DNA-PAINT imaging using FRET-based probes" 17 : 6428-6434, 2017

35 Thompson, M. A., "Extending microscopic resolution with single-molecule imaging and active control" 41 : 321-342, 2012

36 Dempsey, G. T., "Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging" 8 : 1027-, 2011

37 Valley, C. C., "Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer" 26 : 4087-4099, 2015

38 Sochacki, K. A., "Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells" 19 : 352-361, 2017

39 Kim, D. -H., "Direct visualization of single-molecule membrane protein interactions in living cells" 16 : e2006660-, 2018

40 Han, G., "Caged quantum dots" 130 : 15811-15813, 2008

41 Calarese, D. A., "Antibody domain exchange is an immunological solution to carbohydrate cluster recognition" 300 : 2065-2071, 2003

42 Schueder, F., "An order of magnitude faster DNA-PAINT imaging by optimized sequence design and buffer conditions" 16 : 1101-1104, 2019

43 Xu, K., "Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons" 339 : 452-456, 2013

44 Endesfelder, U., "A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment" 141 : 629-638, 2014

45 Wang, Y., "3D super-resolution imaging with blinking quantum dots" 13 : 5233-5241, 2013