CC BY
5DOI 10.24412/cl-37136-2023-1-143-146
SINGLE-MOLECULE LOCALIZATION SUPER-RESOLUTION MICROSCOPY AND ITS
APPLICATIONS
FEN HU1, MENGDI HOU1, FULIN XING1, 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, China 2 State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Responses,
College of Life Sciences, Nankai University, China
ABSTRACT
At the beginning of this century, super-resolution fluorescence microscopy techniques have been invented to break the diffraction limit of traditional optical microscopy, thereby provide unprecedented accesses to life science researches at the micrometer and nanometer scales. Nowadays, a variety of super-resolution techniques have been developed continually, which can be mainly 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]. Owing to the straightforward principle, relatively easy implementation, and outstanding spatial resolution, SMLM has gain more and more attention from researchers.
Single fluorophore labels can be switched "on" and "off" in a controllable manner in SMLM [5]. Afterward, a series of diffraction-limited images featuring each subset of temporarily active and spatially distinct fluorophores can be acquired by repeated imaging sparse stochastic subsets of blinking fluorophores in a sample. 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 image. 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 spatial resolution of the reconstructed SMLM image is determined by the number of photons detected from individual molecules, as well as the labeling efficiency or density [6].
The application of SMLM offers new opportunities to resolve the three-dimensional ultrastructure, organization, and dynamics of subcellular nanostructures at the nanoscale [7]. For instance, dual-color STORM imaging unveils the fantastic periodic structure of membrane-associated skeleton in neuron synapses [8]. STORM clearly resolves the radial eight-fold symmetry structure of nuclear pore complex [9], the radial ninefold symmetry structure of ciliary basal body [10], as well as the nanoscale architecture of necrosomes [11]. STORM also demonstrates the spatial colocalization and functional link of purinosomes with mitochondria [12]. At the meantime, PALM maps the nanoscale protein organization in subcellular molecular assemblies including focal adhesions [13] and podosomes [14]. On the other hand, SMLM have been widely used to measure the nanoscale clustering of membrane receptors such as T cell receptors (TCR), glucose transporter GLUT1 and glutamate receptors (GluR) [15-17]. Besides, SMLM can also be applied in visualizing the dynamic information of cellular organelles and proteins in live cells [18,19].
Utilizing three-dimensional (3D)-STORM super-resolution microscopy, we studied the ultrastructural spatiotemporal organization and regulation of podosome clusters formed in primary mouse peritoneal macrophages (Figure 1) [20]. Podosomes are actin-based protrusive devices critical for cell migration, invasion,
mechanosensing and extracellular matrix remodeling [21]. The unique "core-ring-cap" architecture and highly dynamic natures of podosomes have made them fascinating and attracted many scientists to develop new technologies for their analysis [22]. We first visualized the nanoscale 3D landscape of individual macrophage podosomes with ~20/50 nm (lateral/axial) spatial resolution (Figure 1a). We presented rich information on how podosomes are organized into cluster-like mesoscale superstructures in mouse peritoneal macrophages. 3D-STORM clearly delineates a snowman-like portrait of macrophage podosome core. Then, we performed dual-color 3D-STORM microscopy to reveal the nanoscale mutual localization of the F-actin core, paxillin ring, and myosin IIA ring in the macrophage podosomes. Through a particle superimposing method, we provide the precise mutual localization of podosome actin core and ring component proteins. We thus determined a ~90 nm difference in the axial localization between the paxillin ring and the myosin IIA ring in macrophage podosomes, which is indistinguishable from diffraction-limited microscopy (Figure 1b). Moreover, by dual-color 3D-STORM, we revealed a direct mechanical link between podosomes and microtubules cytoskeletons at the layer of myosin IIA in primary macrophages (Figure 1b). Combined with pharmacological approaches, we suggested that microtubules physically stabilize podosome clusters formed in macrophages in addition to regulate their assembly through the commonly assumed Rho/ROCK-myosin IIA signaling (Figure 1c). These results enriched the information of the nanoscale architecture of podosomes, as well as provide novel insights in the regulation mechanisms of podosomes.
Figure 3 3D-STORM reveals nanoscale architecture and regulation of podosome clusters in primary macrophages. a) The nanoscale 3D landscape of individual podosomes in mouse peritoneal macrophages with different culture time. b) The spatial relationship of podosome components and the crosslink between podosome and microtubules revealed by two-color 3D-STORM. c) Schematic diagram of the mechanisms on microtubules
disruption-caused disassembly of podosomes in macrophages.
Furthermore, by combination of ultrastructure expansion microscopy (U-ExM) with SMLM, we developed a method of U-ExSMLM to resolve the skeleton organization of erythrocytes, a textbook prototype for the submembrane cytoskeleton of metazoan cells [23]. Using poly-l-lysine to treat the glass slide substrate and fix the expanded gel, we realized resistance to shrinkage at the two-dimensional scale, successfully combining ExM and SMLM with a 4.3-fold physical expansion. It increases the 25 nm resolution of SMLM to a molecular resolution of about 6 nm (Figure 2a). 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. Then, we created
weak adhesion conditions that can maintain the native biconcave shape of erythrocytes, and performed imaging with U-ExSMLM. It was found that spectrin skeleton in the erythrocyte dimple region is longer than that in the rim region, while the density of the spectrin skeleton in the dimple region is greater than that in the rim region (Figure 2b). These findings provide direct molecular resolution evidence for the asymmetry of the cytoskeleton in biconcave human erythrocytes. It also provides a novel super-resolution imaging method for studying the subcellular structure and functions near-cytoplasmic membrane.
p-spectrin
f? ..isM^'Hr- • \
\ 'J' 1 .
I J
\ /
■ I U •
"t • 1
* - /
I z depth
Rim Dimple
4 pm
U-ExM Conventional^
Dimple 340 nm <(79 nm)
1
.n.Rrrflil ....
200 300 400 500 Mill 0 100 200 300 400 500 600
tf(nm) cf(nm)
d = distances between nearest neighbors
Rim Dimple
Figure 2 U-ExSMLM achieves molecular resolution. a) Conventional, U-ExM, STORM, and U-ExSMLM images of N-termini of¡3-spectrin of representative erythrocytes. b) U-ExSMLM image of the rim region and dimple region of a biconcave-shaped erythrocyte with N-termini of¡-spectrin labeled. Statistics of density and the peak ofNND (nearest neighboring distance) of the dimple and the rim regions in biconcave erythrocytes.
To sum up, based on SMLM super-resolution microscopy, we elucidate the nanoscale arrangement of podosome clusters in primary peritoneal macrophages with ~20/50 nm (lateral/axial) spatial resolution. Furthermore, we demonstrate that microtubules pass through podosomes at the layer of myosin IIA and physically stabilize podosome clusters in addition to regulate their assembly through the commonly assumed Rho/ROCK-myosin signaling. We also proposed a strategy for the combination of SMLM and ExM relied on poly-l-lysine adhering, reaching the molecular resolution of ~6 nm, and directly revealed the asymmetry of the erythrocyte cytoskeleton, providing a molecular explanation for understanding the unique biconcave morphology in human erythrocytes. These findings will provide new information and innovative methods for the exploration of the ultrastructure and function of subcellular nanostructures.
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. 1178-1188, 2017.
[11] X. Chen, R. Zhu, J. Zhong, Y. Ying, W. Wang, Y. Cao, H. Cai, X. Li, J. Shuai, J. Han, Mosaic composition of RIP1-RIP3 signalling hub and its role in regulating cell death. Nat. Cell Biol. 471-482, 2022.
[12] J. B. French, S. A. Jones, H. Deng, A. M. Pedley, D. Kim, C. Y. Chan, H. Hu, R. J. Pugh, H. Zhao, Y. Zhang, T. J. Huang, Y. Fang, X. Zhuang, S. J. Benkovic, Spatial colocalization and functional link of purinosomes with mitochondria. Science 733-737, 2016.
[13] 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.
[14] 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.
[15] Y. S. Hu, H. Cang, B. F. Lillemeier, Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes. Proc. Natl. Acad. Sci. U. S. A. 7201-7206, 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. Siddig, S. Aufmkolk, S. Doose, M. L. Jobin, C. Werner, M. Sauer, D. Calebiro, Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones. Sci. Adv. eaay7193, 2020.
[18] S. H. Shim, C. Xia, G. Zhong, H. P. Babcock, J. C. Vaughan, B. Huang, X. Wang, C. Xu, G. Q. Bi, X. Zhuang, Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc. Natl. Acad. Sci. U. S. A. 13978-13983, 2012.
[19] 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.
[20] F. Hu, D. Zhu, H. Dong, P. Zhang, F. Xing, W. Li, R. Yan, J. Zhou, K. Xu, L. Pan, J. Xu, Superresolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages. iScience 105514, 2022.
[21] Linder S, Cervero P, Eddy R, et al. Mechanisms and roles of podosomes and invadopodia. Nat Rev Mol Cell Biol 2023, 24: 86-106.
[22] K. van den Dries, S. Linder, I. Maridonneau-Parini, R. Poincloux, Probing the Mechanical Landscape-New Insights Into Podosome Architecture and Mechanics. J. Cell Sci. jcs236828, 2019.
[23] Mengdi Hou, Fulin Xing, Jianyu Yang, Fen Hu, Leiting Pan*, Jingjun Xu. Molecular Resolution Mapping of Erythrocyte Cytoskeleton by Ultrastructure Expansion Single-Molecule Localization Microscopy (U-ExSMLM). Small Methods, 2023, 7: 2201243.
