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多焦点结构光照明显微镜(multifocal structured illumination microscopy, MSIM)能在50 μm的成像深度内实现2倍于衍射极限分辨率的提升, 但在对厚样品成像时, 散射光和离焦光限制了其层析能力和图像衬度. 双光子多焦点结构光照明显微镜(two-photon MSIM, 2P-MSIM)克服了样品组织散射的影响, 进一步提高了MSIM的成像深度和成像特性. 然而, 现有的2P-MSIM通常采用振镜扫描成像, 系统复杂, 灵活性差. 为了解决上述问题, 本文提出了一种基于高速相位型空间光调制器(spatial light modulator, SLM)的双光子多焦点结构光照明超分辨显微成像系统, 通过在SLM上同时加载生成多焦点阵列的相位图和线性相位光栅的相位图, 实现了多焦点阵列的产生和在样品面上的高精度的并行数字随机寻址扫描和激发成像, 结合像素重定位和反卷积技术实现了三维双光子多焦点结构光超分辨成像, 解决了扫描振镜在2P-MSIM成像中的机械惯性问题, 同时降低了系统的复杂性, 提升了灵活性. 在此基础上, 利用搭建的2P-MSIM开展了小鼠肾组织切片和铃兰根茎双光子超分辨成像实验, 验证了该方法的三维超分辨成像能力, 对于2P-MSIM的发展具有重要的意义.
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关键词:
- 多焦点结构光照明显微技术 /
- 双光子 /
- 荧光显微镜 /
- 空间光调制器
Multifocal structured illumination microscopy (MSIM) can achieve a doubled improvement in the resolution of the diffraction limit within an imaging depth of 50 μm. But when imaging thick samples, scattered light and defocused light limit its optical sectioning capability and image contrast. Two-photon MSIM (2P-MSIM) overcomes the influence of sample tissue scattering and further improves the imaging depth and imaging characteristics. However, the existing 2P-MSIM usually adopts galvanometer based scanning mirrors for precisely scanning imaging, which is a complicated and poor flexibility system. Here we propose a simpler 2P-MSIM. Two-photon multifocal scanning imaging can be realized by a spatial light modulator (SLM) with a high frame rate (< 845 Hz). The phase map of generating multi-focus array and linear phase grating loaded on the SLM simultaneously, high-precision parallel digital random address scanning and excitation imaging on the sample surface can be realized. The mechanical inertia problem of the galvanometer scanner in multifocal imaging can be solved by the proposed method while reducing the complexity of the system and improving flexibility. We finally realize two-photon multifocal imaging of mouse kidney tissue slices and lily of the valley rhizome by this system, which verifies the three-dimensional super-resolution imaging capability of this method. It is of great significance in developing the 2P-MSIM.-
Keywords:
- multifocal structured illumination microscopy /
- two-photon /
- fluorescence microscope /
- spatial light modulator
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图 2 (a) WGS算法流程图; (b)得到的相位图; (c)生成的点阵图
Fig. 2. (a) Flow chart of WGS algorithm; (b) generated phase map; (c) generated multi-focus array.
图 3 100 nm荧光珠成像 (a) PinholedWF图像; (b) MPSS图像; (c) MSIM图像; (d)图(a)—(c)中黄色实线所在像素值的大小与对应像素所占宽度的拟合曲线; (e)单个荧光珠的高斯拟合曲线
Fig. 3. 100 nm fluorescent bead imaging: (a) PinholedWF image; (b) MPSS image; (c) MSIM image; (d) fitting curves of the size of the pixel value of the yellow solid lines in panel (a)－(c) vs. the width of corresponding pixel; (e) Gaussian fitting curves of a single fluorescent bead.
图 4 小鼠肾切片的不同厚度层成像图
Fig. 4. Images of different thickness layers of mouse kidney section.
图 5 小鼠肾切片三维成像图 (a) PinholedWF图像; (b) MSIM图像
Fig. 5. Three-dimensional image of mouse kidney section: (a) PinholedWF image; (b) MSIM image.
图 6 铃兰根茎不同厚度层成像图
Fig. 6. Images of different thickness layers of lily of the valley rhizome.
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[1] Hell S W, Wichmann J 1994 Opt. Lett. 19 780
Google Scholar
[2] Betzig E, Patterson G H, Sougrat R, Lindwasser O W, Olenych S, Bonifacino J S, Davidson M W, Lippincott-Schwartz J, Hess H F 2006 Science 313 1642
Google Scholar
[3] Rust M J, Bates M, Zhuang X W 2006 Nat. Methods 3 793
Google Scholar
[4] Gustafsson M G L 2000 J. Microsc. 198 82
Google Scholar
[5] Muller C B, Enderlein J 2010 Phys. Rev. Lett. 104
Google Scholar
[6] York A G, Parekh S H, Nogare D D, Fischer R S, Temprine K, Mione M, Chitnis A B, Combs C A, Shroff H 2012 Nat. Methods 9 749
Google Scholar
[7] Nielsen T, Frick M, Hellweg D, Andresen P 2001 J. Microsc-Oxford 201 368
Google Scholar
[8] Bewersdorf J, Pick R, Hell S W 1998 Opt. Lett. 23 655
Google Scholar
[9] Qu J L, Liu L X, Chen D N, Lin Z Y, Xu G X, Guo B P, Niu H B 2006 Opt. Lett. 31 368
Google Scholar
[10] Ingaramo M, York A G, Wawrzusin P, Milberg O, Hong A, Weigert R, Shroff H, Patterson G H 2014 Proc. Natl. Acad. Sci. U.S.A. 111 5254
Google Scholar
[11] Sacconi L, Froner E, Antolini R, Taghizadeh M R, Choudhury A, Pavone F S 2003 Opt. Lett. 28 1918
Google Scholar
[12] Jureller J E, Kim H Y, Scherer N F 2006 Opt. Express 14 3406
Google Scholar
[13] Shao Y, Qin W, Liu H, Qu J, Peng X, Niu H, Gao B Z 2012 Appl. Phys. B 107 653
Google Scholar
[14] Matsumoto N, Okazaki S, Fukushi Y, Takamoto H, Inoue T, Terakawa S 2014 Opt. Express 22 633
Google Scholar
[15] Matsumoto N, Konno A, Ohbayashi Y, Inoue T, Matsumoto A, Uchimura K, Kadomatsu K, Okazaki S 2017 Opt. Express 25 7055
Google Scholar
[16] Sinclair G, Leach J, Jordan P, Gibson G, Yao E, Laczik Z J, Padgett M J, Courtial J 2004 Opt. Express 12 1665
Google Scholar
[17] Curtis J E, Koss B A, Grier D G 2002 Opt. Commun. 207 169
Google Scholar
[18] Meister M, Winfield R J 2002 Opt. Commun. 203 39
Google Scholar
[19] Di Leonardo R, Ianni F, Ruocco G 2007 Opt. Express 15 1913
Google Scholar
[20] Kim D, Keesling A, Omran A, Levine H, Bernien H, Greiner M, Lukin M D, Englund D R 2019 Opt. Lett. 44 3178
Google Scholar
[21] Sheppard C J R, Gu M 1990 Opitk 86 104
Google Scholar
[22] Roider C, Heintzmann R, Piestun R, Jesacher A 2016 Opt. Express 24 15456
Google Scholar
[23] Schmitz C H J, Spatz J P, Curtis J E 2005 Opt. Express 13 8678
Google Scholar
[24] Li S W, Wu J J, Li H, Lin D Y, Yu B, Qu J L 2018 Opt. Express 26 23585
Google Scholar
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