PDF(1536 KB)
基于谐波的多光子扫描结构光照明超分辨显微成像
Lei Wang, Xiaomin Zheng, Jie Zhou, Meiting Wang, Jiajie Chen, Youjun Zeng, Gaixia Xu, Ying Wang, Haixia Qiu, Yonghong Shao, Junle Qu, Bruce Zhi Gao, Ying Gu
工程（英文） ›› 2022, Vol. 16 ›› Issue (9) : 65-72.
PDF(1536 KB)
PDF(1536 KB)
基于谐波的多光子扫描结构光照明超分辨显微成像
Improvement in Resolution of Multiphoton Scanning Structured Illumination Microscopy via Harmonics
本文提出了一种利用多光子非线性效应的多光子结构光照明超分辨显微成像（multiphoton-structured illumination microscopy, mP-SIM）技术，可实现比线性SIM 更高的成像分辨率。通过扫描正弦结构的照明光激发样本产生多光子荧光信号或谐波信号，从而利用由光学非线性效应产生的非正弦结构信号光的谐波来提高分辨率。本文提出了mP-SIM 理论以重建系统的超分辨图像。如果考虑多光子非线性效应的所有高阶谐波（阶数足够高，即m足够大），则mP-SIM 在理论上可实现无限分辨率。通过实验证实了双光子荧光（2P）-SIM 和二次谐波产生（second harmonic generation, SHG）-SIM 分别可实现86 nm 和72 nm的横向分辨率。对细胞内染色F-肌动蛋白和小鼠尾腱胶原纤维成像，进一步验证了mP-SIM 的超分辨成像能力。该方法不但适用于多光子荧光超分辨显微成像，而且适用于非标记的如SHG等超分辨显微成像；重要的是，该方法不需要特定的荧光团或高功率激发光，可直接用于商用mP显微系统来实现超分辨成像。
We describe a multiphoton (mP)-structured illumination microscopy (SIM) technique, which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence. This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal. The harmonics of the structured fluorescence illumination are utilised to improve resolution. We present an mP-SIM theory for reconstructing the super-resolution image of the system. Theoretically, the resolution of our mP-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered. Experimentally, we demonstrate an 86-nm lateral resolution for 2P-SIM and a 72-nm lateral resolution for second-harmonic-generation (SHG)-SIM. We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon. Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.
超分辨显微成像 / 结构光照明超分辨显微成像 / 多光子结构光照明超分辨显微成像 / SIM 二次谐波产生（SHG）-SIM /
Super-resolution microscopy / Structured illumination microscopy / Multiphoton structured illumination microscopy / SIM
| [1] |
Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science 1990;248(4951):73–6.
|
| [2] |
Centonze VE, White JG. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 1998;75(4):2015–24.
|
| [3] |
Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat Methods 2005;2(12):932–40.
|
| [4] |
Konig K. Multiphoton microscopy in life sciences. J Micros 2000;200(2): 83–104.
|
| [5] |
Ingaramo M, York AG, Wawrzusin P, Milberg O, Hong A, Weigert R, et al. Twophoton excitation improves multifocal structured illumination microscopy in thick scattering tissue. Proc Natl Acad Sci USA 2014;111(14):5254–9.
|
| [6] |
Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 2006;313(5793):1642–5.
|
| [7] |
Hess ST, Girirajan TPK, Mason MD. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 2006;91(11): 4258–72.
|
| [8] |
Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 2006;3(10): 793–6.
|
| [9] |
Bates M, Huang Bo, Dempsey GT, Zhuang X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 2007;317(5845): 1749–53.
|
| [10] |
Huang B, Wang W, Bates M, Zhuang X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 2008;319(5864): 810–3.
|
| [11] |
Hell SW, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission–depletion fluorescence microscopy. Opt Lett 1994;19(11):780–2.
|
| [12] |
Klar TA, Hell SW. Subdiffraction resolution in far-field fluorescence microscopy. Opt Lett 1999;24(14):954–6.
|
| [13] |
Gustafsson MGL. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Micros 2000;198(2): 82–7.
|
| [14] |
Heintzmann R, Crème CG. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. In: Proceedings of Optical Biopsies and Microscopic Techniques III; 1998 Sep 8–12; Stockholm, Sweden. SPIE; 1998. p. 185–96.
|
| [15] |
Huang B, Babcock H, Zhuang X. Breaking the diffraction barrier: superresolution imaging of cells. Cell 2010;143(7):1047–58.
|
| [16] |
Sahl SJ, Hell SW, Jakobs S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 2017;18(11):685–701.
|
| [17] |
Eggeling C, Willig KI, Sahl SJ, Hell SW. Lens-based fluorescence nanoscopy. Q Rev Biophys 2015;48(2):178–243.
|
| [18] |
Kolmakov K, Wurm CA, Meineke DNH, Göttfert F, Boyarskiy VP, Belov VN, et al. Polar red-emitting rhodamine dyes with reactive groups: synthesis, photophysical properties, and two-color STED nanoscopy applications. Chem Eur J 2014;20(1):146–57.
|
| [19] |
Schmidt R, Wurm CA, Jakobs S, Engelhardt J, Egner A, Hell SW. Spherical nanosized focal spot unravels the interior of cells. Nat Methods 2008;5(6): 539–44.
|
| [20] |
Westphal V, Hell SW. Nanoscale resolution in the focal plane of an optical microscope. Phys Rev Lett 2005;94(14):143903.
|
| [21] |
Gustafsson MGL. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 2005;102(37):13081–6.
|
| [22] |
Heintzmann R, Gustafsson MGL. Subdiffraction resolution in continuous samples. Nat Photonics 2009;3(7):362–4.
|
| [23] |
Rego EH, Shao L, Macklin JJ, Winoto L, Johansson GA, Kamps-Hughes N, et al. Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50 nm resolution. Proc Natl Acad Sci USA 2012;109(3):E135–43.
|
| [24] |
Li D, Shao L, Chen BC, Zhang X, Zhang M, Moses B, et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 2015;349(6251):aab3500.
|
| [25] |
Kner P, Chhun BB, Griffis ER, Winoto L, Gustafsson MGL. Super-resolution video microscopy of live cells by structured illumination. Nat Methods 2009;6(5):339–42.
|
| [26] |
Fiolka R, Shao L, Rego EH, Davidson MW, Gustafsson MGL. Time-lapse twocolor 3D imaging of live cells with doubled resolution using structured illumination. Proc Natl Acad Sci USA 2012;109(14):5311–5.
|
| [27] |
Shao L, Kner P, Rego EH, Gustafsson MGL. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods 2011;8(12): 1044–6.
|
| [28] |
Dan D, Lei M, Yao B, Wang W, Winterhalder M, Zumbusch A, et al. DMD-based LED-illumination super-resolution and optical sectioning microscopy. Sci Rep 2013;3(1):1116.
|
| [29] |
Swayne T, Zhou C, Boldogh I, Charalel J, McFaline-Figueroa J, Thoms S, et al. Role for cER and Mmr1p in anchorage of mitochondria at sites of polarized surface growth in budding yeast. Curr Biol 2011;21(23):1994–9.
|
| [30] |
Olshausen PV, Defeu Soufo HJ, Wicker K, Heintzmann R, Graumann PL, Rohrbach A. Superresolution imaging of dynamic MreB filaments in B. subtilis— a multiple-motor-driven transport? Biophys J 2013;105(5):1171–81.
|
| [31] |
Wheeler R, Mesnage S, Boneca IG, Hobbs JK, Foster SJ. Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria. Mol Microbiol 2011;82(5):1096–109.
|
| [32] |
Markaki Y, Smeets D, Fiedler S, Schmid VJ, Schermelleh L, Cremer T, et al. The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture. BioEssays 2012;34(5):412–26.
|
| [33] |
Fitzgibbon J, Bell K, King E, Oparka K. Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy. Plant Physiol 2010;153(4):1453–63.
|
| [34] |
Sonnen KF, Schermelleh L, Leonhardt H, Nigg EA. 3D-structured illumination microscopy provides novel insight into architecture of human centrosomes. Biol Open 2012;1(10):965–76.
|
| [35] |
Bianchini P, Harke B, Galiani S, Vicidomini G, Diaspro A. Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging. Proc Natl Acad Sci USA 2012;109(17):6390–3.
|
| [36] |
Lu J, Min W, Conchello JA, Xie XS, Lichtman JW. Super-resolution laser scanning microscopy through spatiotemporal modulation. Nano Lett 2009;9(11):3883–9.
|
| [37] |
Urban BE, Yi J, Chen S, Dong B, Zhu Y, DeVries SH, et al. Super-resolution twophoton microscopy via scanning patterned illumination. Phys Rev E Stat Nonlinear Soft Matter Phys 2015;91(4):042703.
|
| [38] |
Winter PW, York AG, Nogare DD, Ingaramo M, Christensen R, Chitnis A, et al. Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples. Optica 2014;1(3):181–91.
|
| [39] |
Cheng LC, Horton NG, Wang K, Chen SJ, Xu C. Measurements of multiphoton action cross sections for multiphoton microscopy. Biomed Opt Express 2014;5(10):3427–33.
|
| [40] |
Wicker K, Heintzmann R. Resolving a misconception about structured illumination. Nat Photonics 2014;8(5):342–4.
|
| [41] |
Bianchini P, Diaspro A. Three-dimensional (3D) backward and forward second harmonic generation (SHG) microscopy of biological tissues. J Biophotonics 2008;1(6):443–50.
|
| [42] |
Fujita K, Kobayashi M, Kawano S, Yamanaka M, Kawata S. High-resolution confocal microscopy by saturated excitation of fluorescence. Phys Rev Lett 2007;99(22):228105–9.
|
/
| 〈 |
|
〉 |
