CC BY
8SINGLE-MOLECULE LOCALIZATION SUPER-RESOLUTION MICROSCOPY AND ITS APPLICATIONS FEN HU1, MENGDI HOU1, JIANYU YANG1, AND LEITING PAN1,2
1The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied
Physics, Nankai University, Tianjin 300071, China 2 State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Responses, College of Life Sciences, Nankai
University, Tianjin 300071, China
plt@nankai.edu.cn
ABSTRACT
Over the past two decades, a batch of super-resolution fluorescence microscopy methods have been developed to break the diffraction limit of traditional optical microscopy, thereby greatly improves the study of subcellular architectures and dynamics in biological systems. Generally, these super-resolution techniques can be divided into two classes: (1) Patterned illumination-based microscopy involving stimulated emission depletion microscopy (STED) [1] and structured illumination microscopy (SIM) [2]; (2) Single-molecule localization microscopy (SMLM) such as photoactivated localization microscopy (PALM) [3] and stochastic optical reconstruction microscopy (STORM) [4]. Due to the straightforward principle, relative ease of implementation, and unmatched spatial resolution than other super-resolution imaging techniques, SMLM has become particularly popular for many researchers.
In SMLM, single fluorophore labels can be switched "on" and "off" in a controllable manner [5]. By repeated imaging sparse stochastic subsets of blinking fluorophores in a sample, a series of diffraction-limited images featuring each subset of temporarily active and spatially distinct fluorophores can be acquired. Then, individual fluorophores are localized at sub-diffraction precision by finding the centers of their point spread functions. Finally, the superposition of the calculated particle positions will generate a super-resolution reconstruction. Based on the sparse fluorophore detection, localization and superposition, SMLM can discern and pinpoint individual molecules even with dense labeling, thus allow molecular localization accuracies at the nanometer level. The number of photons detected from individual molecules, the labeling efficiency or density are important factors determining the spatial resolution of SMLM image [6].
Nowadays SMLM has become a powerful tool to study the three-dimensional ultrastructure and organization of subcellular nanostructures [7]. For instance, dual-color STORM imaging enables the discovery of the beautiful membrane-associated periodic skeleton in synapses of neurons [8]. STORM also clearly resolves the radial eight-fold symmetry of nuclear pore scaffold structure [9] and radial nine-fold symmetry of centriole-containing complex [10], as well as the delicate structure of ESCRT-III complex during abscission of the intercellular bridge connecting two dividing cells [11]. Meanwhile, PALM maps the nanoscale protein organization in molecular assemblies including focal adhesions [12] and podosomes [13]. On the other hand, SMLM have been widely used to measure the nanoscale clustering of membrane receptors such as EGFR, ryanodine receptors (RyR) and GLUT1 [14-16]. Besides, SMLM has also enhanced our ability in visualizing the dynamic information of cellular organelles and proteins in live cells [17,18].
Utilizing STORM super-resolution microscopy, we resolved the native ultrastructure of erythrocyte cytoskeleton [19], a textbook prototype for the submembrane cytoskeleton of metazoan cells. By STORM imaging of the N termini of P-spectrin, we determined an ~80-nm junction-to-junction distance, a length consistent with relaxed spectrin tetramers and theories based on spectrin abundance (Fig. 1). While early experiments suggest a triangular network of actin-based junctional complexes connected by ~200-nm-long spectrin tetramers, later studies indicate much smaller junction-to-junction distances in the range of 25-60 nm. Our results call for a reassessment of the structure and function of the submembrane cytoskeleton.
Figure 1 STORM reveals native ultrastructure of the erythrocyte membrane skeleton
In addition, by combination of ultrastructure expansion microscopy (U-ExM) with SMLM, we developed a method of U-ExSMLM to resolve the skeleton organization of erythrocytes at molecular resolution (Fig. 2). We investigated the nanoscale spatial distribution of several skeleton proteins, including N/C-terms of p-spectrin, protein 4.1, as well as tropomodulin. Our results by U-ExSMLM were in good accordance with the acknowledged model of erythrocyte skeleton structure.
_ * ; ' * i j*
0 2 um ém V ;' □ 1-1 % O . V ; 1 200 nm • • . • V" »* « •
V, conventional STORM- * : / ' « • • ** *M , L0 • • ^ _ ^ ^ • _ • i .'. ,1
... V»^ . • *
dfZ ■. i'V'iV ë' ' {i ' .: A:.' '. v ■ ' '. '800 nm
; - ! V / • • . * *
z ' . \ "■' . -i V■ : Ç . . V _ ., ... -t ' ;. .- \ ■ ■ : ■ -:'VJ >>;•'■ ■■ ■ .■ ■ - • * « * t ' » .
U-ExM" U Hx'SMhM U-ExSMLM ,« • • <
Figure 2 U-ExSMLM resolves the organization of erythrocyte membrane skeleton at molecular resolution
REFERENCES
[1] T. A. Klar, S. Jakobs, M. Dyba, A. Egner, S. W. Hell, Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. U. S. A. 8206-8210, 2000.
[2] M. G. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. U. S. A. 13081-13086, 2005.
[3] E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, Imaging intracellular fluorescent proteins at nanometer resolution. Science 1642-1645, 2006.
[4] M. J. Rust, M. Bates, X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 793-796, 2006.
[5] K. Finan, B. Flottmann, M. Heilemann, Photoswitchable fluorophores for single-molecule localization microscopy. Methods Mol. Biol. 131-151, 2013.
[6] R. Diekmann, M. Kahnwald, A. Schoenit, J. Deschamps, U. Matti, J. Ries, Optimizing imaging speed and excitation intensity for single-molecule localization microscopy. Nat. Methods 909-912, 2020.
[7] Y. M. Sigal, R. Zhou, X. Zhuang, Visualizing and discovering cellular structures with super-resolution microscopy. Science 880-887, 2018.
[8] K. Xu, G. Zhong, X. Zhuang, Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science
452-456, 2013.
[9] A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, J. Ellenberg, Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 655-658, 2013.
[10] X. Shi, G. Garcia 3rd, J. C. Van De Weghe, R. McGorty, G. J. Pazour, D. Doherty, B. Huang, J. F. Reiter, Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome. Nat. Cell Biol. 11781188, 2017.
[11] I. Goliand, S. Adar-Levor, I. Segal, D. Nachmias, T. Dadosh, M. M. Kozlov, N. Elia, Resolving ESCRT-III Spirals at the Intercellular Bridge of Dividing Cells Using 3D STORM. Cell Rep. 1756-1764, 2018.
[12] P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, C. M. Waterman, Nanoscale architecture of integrin-based cell adhesions. Nature 580-584, 2010.
[13] J. C. Herron, S. Hu, T. Watanabe, A. T. Nogueira, B. Liu, M. E. Kern, J. Aaron, A. Taylor, M. Pablo, T. L. Chew, T. C. Elston, K. M. Hahn, Actin nano-architecture of phagocytic podosomes. Nat. Commun. 4363, 2022.
[14] J. Gao, Y. Wang, M. Cai, Y. Pan, H. Xu, J. Jiang, H. Ji, H. Wang, Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging. Nanoscale 2511-2519, 2015.
[15] I. Jayasinghe, A. H. Clowsley, R. Lin, T. Lutz, C. Harrison, E. Green, D. Baddeley, L. Di Michele, C. Soeller, True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors. Cell Rep. 557-567, 2018.
[16] Q. Yan, Y. Lu, L. Zhou, J. Chen, H. Xu, M. Cai, Y. Shi, J. Jiang, W. Xiong, J. Gao, H. Wang, Mechanistic insights into GLUT1 activation and clustering revealed by super-resolution imaging. Proc. Natl. Acad. Sci. U. S. A. 7033-7038, 2018.
[17] S. H. Shim, C. Xia, G. Zhong, H. P. Babcock, J. C. Vaughan, B. Huang, X. Wang, C. Xu, G. Q. Bi, X. Zhuang, Superresolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc. Natl. Acad. Sci. U. S. A. 13978-13983,2012.
[18] L. Xiang, K. Chen, R. Yan, W. Li, K. Xu, Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity. Nat. Methods 524-530, 2020.
[19] L. Pan, R. Yan, W. Li, K. Xu, Super-resolution microscopy reveals the native ultrastructure of the erythrocyte cytoskeleton. Cell Rep. 1151-1158, 2018.
