-
由于微球直径通常要求在微米级别,与探测光的波长量级相同,光在微球上的散射效应无法忽略。通常认为,经典几何光学理论在这一尺度上关于焦点、焦距和放大率等理论并不准确,无法合理揭示微球透镜显微成像的机理。然而,新发展起来的倏逝波传输理论和光子纳米喷流效应一定程度上解释了衍射极限的突破现象,为微透镜超分辨显微成像提供了理论基础。
-
物体受光波照射后,离开表面的光波分为两种:一部分光向远方传播,被传统光学显微镜接收;另一部分光波只能沿物体表面传播,一旦离开表面就很快衰减,这部分光被称作倏逝波。倏逝波携带物体的高频图像信息,但在一般情况下无法被显微物镜接收。微球透镜能把近场的倏逝波耦合进入内部,将其转变成可以在远场传播的传播波并被物镜接收。在微球透镜中会激发回音壁模式(Whispering gallery mode, WGM),分辨率可以通过WGM增强而超过衍射极限[9]。
Ben-Aryeh Y[10-12]对微球透镜把倏逝波转变成传播波的条件及过程进行了分析,如图1所示,设入射光波的水平波数为
$u$ ,垂直波数为$w$ ,则介质和微球分界面P处入射波的水平分量为:$$ {u}'=\sqrt{{\left(u{\rm{cos}}\theta \right)}^{2}-{\left(\right|w\left|{\rm{sin}}\theta \right)}^{2}} $$ 当水平分量小于微球内部所允许传播的最大波数时,将会转化为传播波,即:
${{u}}' < {{{{n}}}}{{{{{{k}}}}}}_{0}$ ;为了避免微球透镜内部的传播波无法传递到远场, 水平分量应小于周围浸没介质的波数,即:${{u}}' < {{{{n}}}}_{{{{l}}}}{{{{k}}}}_{0}$ 。其中,$ n $ 为微球折射率,$ {n}_{l} $ 为微球周围浸没介质折射率,$ {k}_{0} $ 为单位长度上产生的波数变化,即波数。理论上,提高微球透镜的折射率
$ n $ 和周围浸没介质的折射率$ {n}_{l}, $ 则微球耦合的高频成分增加,成像的分辨率提升。由于倏逝波竖直方向快速衰减的特点,微球透镜耦合近场光学信息受到高度限制,仅能将距离样品表面一定高度范围内的倏逝波加以耦合并将其传递至远场。 -
平行(或称准直)光束经介电圆柱或者微球聚焦,在其背光面会出现一个喷射长度大于波长尺寸,喷射光束宽度处于亚波长量级的超强聚焦光场,这个现象称为光子纳米喷流效应。Mie理论对球形粒子光散射进行了完整描述,其基于麦克斯韦方程组,可以严格求出在平面波照射下,微粒在均匀且各向同性的介质中,散射场及内场的精确解。即可给出光子纳米喷流效应的形成,从而构建系统电磁场的分布情况,进一步对其性质进行研究[13]。微球透镜和传统透镜所形成的焦点类似,但是其形成机理与性质却不相同,形成光子纳米喷流的位置与微透镜类型和周围介质的折射率有关。从聚焦的角度分析,微球产生的纳米喷流越细,微球的聚焦效果越好,分辨率越高[14]。
光子纳米喷射需要用一些参数来表征。如图2所示,喷射光强最大的点即为聚焦点,从微球的球心到聚焦点的距离为微球系统的焦距( f )。喷射长度(zr)定义为从光强最大值(Imax)点到沿轴方向光强衰减到
$\dfrac{{{I_{{\rm{max}}}}}}{{{\rm{e}^2}}}$ 那一点的距离。半最大值全宽(FWHM)指在光强最大值位置处,光强最大值一半的横向距离。半最大值全宽被广泛应用于评价超分辨性能,其值越小,即束腰(w0)的宽越窄,超分辨能力越强。普遍认为,当光强越大、焦距越短、纳米喷流的纵向延续长度越长、半高全宽越小时,超分辨显微成像效果越好。且焦点位置在微球外内影响超分辨显微成像虚实[15]。微球超透镜成像的放大倍率、分辨率等性质参数与光子纳米喷射表征参数有一定联系。光子纳米射流的束腰和微球与最大电强度位置之间的距离决定了超分辨显微成像的能力,并随微球的折射率和尺寸发生变化[16]。微球的尺寸减小时,纳米射流的束腰也减小。纳米射流形成在靠近微球表面时,有利于提高分辨率和放大率[17]。由于光子纳米射流现象既超出了近场光学范畴(一个波长以内),又不属于传统远场光学(毫米量级及以上),因此其是一种准近场光学现象,所以只有当观测样品与介质微球之间的距离在约20个波长以内时,微球才具有超分辨能力。同时以光子射流热点区域为焦点,可以推断出图像的性质(实像和虚像),及像面位置和微球放大率的趋势[18]。
Progress in microspheric lens based super-resolution microscopic imaging technology with large field of view
-
摘要: 光学显微镜是人类探索微观世界的重要工具,在生物学、医学、材料学、精密测量学等领域发挥重要作用。由于衍射极限的存在,发展更高质量、更高空间分辨率的超分辨光学显微成像技术成为当下研究的前沿热点。基于微球透镜的超分辨显微成像技术有着易于实现、简单直接和免标记的显著优点,发展潜力巨大。但是单个微球的视野有限,且难以进行精确定位。提高微球的可操控性,拓展超分辨显微成像视场的范围,已成为该技术突破发展的核心关键。文中在介绍微球超分辨的成像原理,分析影响成像质量主要因素的基础上,重点总结了国内外团队在拓展微球透镜超分辨显微成像视场方面的最新研究进展。根据微球的操控方式,将研究工作总结为机械接触控制、微球辅助增强层、非接触控制和微球物镜一体化四类进行介绍,探讨其技术特点,并对大视场成像、图像拼接等面向视场拓展的图像处理技术进行论述。最后,提出微球透镜超分辨显微成像技术亟待解决的关键问题、存在的难点与挑战,以及未来开展研究工作的突破点,展望了该技术的发展与应用拓展方向。Abstract: Optical microscope is a vital tool to explore the microscopic world for humans, which plays an important role in the fields of biology, medicine, materials science, and precision measurement. Due to the diffraction limit, developing super-resolution optical microscopy imaging technology with higher image quality and spatial resolution has become a hot research frontier. Super-resolution imaging technology based on microspheric lens has great development potential because it's obvious advantages of being easy to implement, simple operation and label-free. However, the field of view (FOV) of a single microsphere is limited, and it is difficult to locate the microspheres accurately. Improving the maneuverability of microspheres and expanding the FOV of super-resolution imaging have become the key of this technology development. Based on the principle of microsphere super-resolution imaging technology and the main factors for imaging quality, the paper focuses on the latest research progress in expanding the FOV of microspheric lens super-resolution microscopy imaging. According to the control methods of the microsphere, these progresses are summarized into four categories: Mechanically contact control, non-contact control, microsphere assembly layer, and microsphere-objective integration. The technical characteristics of these four categories are discussed, and the image processing technologies for field expansion are also analyzed, such as large FOV and image stitching. At the end, the paper points out the key problems, existing difficulties and challenges for microspheric lens super-resolution imaging technology, as well as the breakthrough for the future research work. The development direction and application future of this technology are prospected.
-
图 4 (a1) 纳钙玻璃微球成像;(a2) 钛酸钡微球成像[20] ;(b1)、(b2) 和(b3) 分别是400 nm周期物体在30 μm、55 μm和90 μm Z位置处的虚像,比例尺:1 μm
Figure 4. (a1) Soda lime glass microspheres imaging; (a2) Barium titanate glass microspheres imaging [20]; (b1), (b2) and (b3) are the virtual images of the 400 nm periodic object at Z positions of 30 μm, 55 μm and 90 μm, respectively. Scale bars represent 1 μm
图 9 机械接触控制类装置示意图和超分辨成像图。(a)、(e)毛细玻璃管法[32];(b)、(f)悬臂梁法[33];(c)、(g) ST型钨探针法[34];(d)、(h)AFM探针法[36]
Figure 9. Schematic diagram and super-resolution images of mechanical strut devices. (a),(e) Capillary glass tube method [32]; (b),(f) Cantilever beam method [33]; (c),(g) ST-type tungsten probe method [34]; (d),(h) AFM probe method [36]
图 11 (a) 纳米游泳机器人示意图;(b) 化学动力推进示意图;(c) 涂覆示意图;(d) SEM图;(e) 红色箭头显示微型机器人的扫描运动;(f) 拼接视频单帧的放大区域得到图像[39]
Figure 11. (a) Schematic illustration of swimming microrobot optical nanoscopy (SMON); (b) Schematic illustration of the chemically powered propulsion; (c) Schematic diagram of coating; (d) SEM image; (e) Tracking line showing the motion of a microrobot scanning; (f) Image by stitching the magnified area from individual video frames[39]
图 12 (a) 周期为278 nm、线宽为139 nm 的硅纳米结构光栅(SNG)的SEM 图像;(b)被捕获的PS球对SNG成像;(c)被捕获的MF球对SNG成像[41]
Figure 12. (a) The SEM image of the silicon nanostructure grating (SNG) with a period of 278 nm and a 139 nm line-width; (b) The SNG image by the trapped PS sphere; (c) The SNG image assisted by a trapped MF sphere[41]
图 13 (a)光学正置显微镜;(b) Z轴平移器;(c)夹住玻璃微球阵列芯片的定制铝框[45];(d)微球阵列芯片;(e)微球阵列芯片制作工艺示意图[53]; (f)超透镜通过透镜适配器组合到传统显微物镜上[48];(g) PCM镜头的制备和安装[49]
Figure 13. (a) Optical upright microscope; (b) Z-axis translator; (c) A custom aluminum frame for clamping the glass microsphere array chip[45]; (d) Fabricated microsphere array chip; (e) Schematic of the fabrication process of the microsphere array chip[53]; (f) Super objective was made by integrating a conventional microscope objective lens using a adaptor [48]; (g) Preparation of PCM lens and installation [49]
-
[1] Abbe E. Beitrge zur theorie des mikroskops und der mikroskopischen wahrnehmung [J]. Archiv für Mikroskopische Anatomie, 1873, 9(1): 413-468. [2] Hell S W, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy [J]. Optics Letters, 1994, 19(11): 780-782. doi: 10.1364/OL.19.000780 [3] Betzig E, Patterson G H, Sougratr, et al. Imaging intracellular fluorescent proteins at nanometer resolution [J]. Science, 2006, 1642(313): 1127344. doi: 10.1126/science.1127344 [4] Gustafsson M G L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy [J]. Journal of Microscopy, 2000, 198(2): 82-87. doi: 10.1046/j.1365-2818.2000.00710.x [5] Ebbesen T W, Lezec H J, Ghaemi H F, et al. Extraordinary optical transmission through sub-wavelength hole arrays [J]. Nature Materials, 2010(5S): 35-37. doi: 10.1038/35570 [6] Chen Zhigang, Allen T, Vadim B. Photonic nanojet enhancement of backscattering of light by nanoparticles: A potential novel visible-light ultramicroscopy technique [J]. Optics Express, 2004, 12(7): 1214-1220. [7] Wang Z, Guo W, Li L, et al. Optical virtual imaging at 50 nm lateral resolution with a white-light nanoscope [J]. Nature Communications, 2011, 2(1): 1-6. [8] Perrin S, Li H, Lecler S, et al. Unconventional magnification behaviour in microsphere-assisted microscopy [J]. Optics & Laser Technology, 2019, 114: 40-43. [9] Duan Y, Barbastathis G, Zhang B. Classical imaging theory of a microlens with super-resolution [J]. Optics Letters, 2013, 38(16): 2988-2990. doi: 10.1364/OL.38.002988 [10] Ben-Aryeh Y. Tunneling of evanescent waves into propagating waves [J]. Applied Physics B, 2006, 84(1/2): 121-124. [11] Ben-Aryeh Y. Transmission enhancement by conversion of evanescent waves into propagating waves [J]. Applied Physics B, 2008, 91(1): 157-165. doi: 10.1007/s00340-008-2945-2 [12] Ben-Aryeh Y. Superresolution observed from evanescent waves transmitted through nano-corrugated metallic films [J]. Applied Physics B, 2012, 109(1): 165-170. doi: 10.1007/s00340-012-5193-4 [13] Lukiyanchuk B S, Paniagua-Domínguez R, Minin I, et al. Refractive index less than two: photonic nanojets yesterday, today and tomorrow [J]. Optical Materials Express, 2017, 7(6): 1820-1847. doi: 10.1364/OME.7.001820 [14] Yang H, Trouillon R, Huszka G, et al. Super-resolution imaging of a dielectric microsphere is governed by the waist of its photonic nanojet [J]. Nano Letters, 2016, 16(8): 4862-4870. doi: 10.1021/acs.nanolett.6b01255 [15] Devilez A, Stout B, Bonod N, et al. Spectral analysis of three-dimensional photonic jets [J]. Optics Express, 2008, 16(18): 14200-14212. doi: 10.1364/OE.16.014200 [16] Lee S, Li L, Wang Z. Optical resonances in microsphere photonic nanojets [J]. Journal of Optics, 2014, 16(1): 5704. doi: 10.1088/2040-8978/16/1/015704 [17] Yang H, Gijs M A M. Optical microscopy using a glass microsphere for metrology of sub-wavelength nanostructures [J]. Microelectronic Engineering, 2015, 143: 86-90. doi: 10.1016/j.mee.2015.03.072 [18] Lecler S, Perrin S, Leong-Hoi A, et al. Photonic jet lens [J]. Scientific reports, 2019, 9(1): 1-8. [19] Darafsheh A, Walsh G F, Negro L D, et al. Optical super-resolution by high-index liquid-immersed microspheres [J]. Applied Physics Letters, 2012, 101(14): 388-457. [20] Darafsheh A, Limberopoulos N I, Derov J S, et al. Advantages of microsphere-assisted super-resolution imaging technique over solid immersion lens and confocal microscopies [J]. Applied Physics Letters, 2014, 104(6): 061117. doi: 10.1063/1.4864760 [21] Lee S, Li L, Ben-Aryeh Y, et al. Overcoming the diffraction limit induced by microsphere optical nanoscopy [J]. Journal of Optics, 2013, 15(12): 125710. doi: 10.1088/2040-8978/15/12/125710 [22] Lee S, Li L, Wang Z, et al. Immersed transparent microsphere magnifying sub-diffraction-limited objects [J]. Applied Optics, 2013, 52(30): 7265-7270. doi: 10.1364/AO.52.007265 [23] Li L, Guo W, Yan Y, et al. Label-free super-resolution imaging of adenoviruses by submerged microsphere optical nanoscopy [J]. Light: Science & Applications, 2013, 2(9): e104. [24] Hao X, Kuang C, Liu X, et al. Microsphere based microscope with optical super-resolution capability [J]. Applied Physics Letters, 2011, 99(20): 203102. doi: 10.1063/1.3662010 [25] Zhou Y, Tang Y, He Y, et al. Effects of immersion depth on super-resolution properties of index-different microsphere-assisted nanoimaging [J]. Applied Physics Express, 2018, 11(3): 032501. doi: 10.7567/APEX.11.032501 [26] Zhou Y, Tang Y, Deng Q, et al. Contrast enhancement of microsphere-assisted super-resolution imaging in dark-field microscopy [J]. Applied Physics Express, 2017, 10(8): 082501. doi: 10.7567/APEX.10.082501 [27] Zhou J, Zeng B, Bi S, et al. Enhanced magnification factors in super-resolution imaging using stacked dual microspheres [J]. Journal of Optics, 2020, 22(8): 085605. doi: 10.1088/2040-8986/aba03c [28] Luo H, Yu H, Wen Y, et al. Enhanced high-quality super-resolution imaging in air using microsphere lens groups [J]. Optics Letters, 2020, 45(11): 2981-2984. doi: 10.1364/OL.393041 [29] Guo M, Ye Y H, Hou J, et al. Imaging of sub-surface nanostructures by dielectric planer cavity coupled microsphere lens [J]. Optics Communications, 2017, 383: 153-158. doi: 10.1016/j.optcom.2016.09.002 [30] Yang S, Cao Y, Shi Q, et al. Label-free super-resolution imaging of transparent dielectric objects assembled on silver film by a microsphere-assisted microscope [J]. The Journal of Physical Chemistry C, 2019, 123(46): 28353-28358. doi: 10.1021/acs.jpcc.9b07285 [31] Shi Q F, Yang S L, Cao Y R, et al. Super-resolution imaging of low-contrast periodic nanoparticle arrays by microsphere-assisted microscopy [J]. Chinese Physics B, 2021, 30(4): 040702. doi: 10.1088/1674-1056/abcf48 [32] Krivitsky L A, Wang J J, Wang Z, et al. Locomotion of microspheres for super-resolution imaging [J]. Scientific Reports, 2013, 3(1): 1-5. [33] Wang S, Zhang D, Zhang H, et al. Super-resolution optical microscopy based on scannable cantileverʜcombined microsphere [J]. Microscopy Research and Technique, 2015, 78(12): 1128-1132. doi: 10.1002/jemt.22595 [34] Meng K, Gao S, Zhang Y, et al. Optical super-resolution imaging study based on controlling liquid-immersed microsphere[C]//2018 IEEE 13th Annual International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). IEEE, 2018: 538-542. [35] Meng Kai. Research on microsphere lens operating system for super-resolution optical imaging[D]. Suzhou: Soochow University, 2019. (in Chinese) [36] Wang F, Liu L, Yu H, et al. Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging [J]. Nature Communications, 2016, 7(1): 1-10. [37] Allen K W, Farahi N, Li Y, et al. Super-resolution imaging by arrays of high-index spheres embedded in transparent matrices[C]//Naecon 2014-IEEE National Aerospace and Electronics Conference. IEEE, 2014: 50-52. [38] Allen K W, Farahi N, Li Y, et al. Super-resolution microscopy by movable thin-films with embedded microspheres: Resolution analysis [J]. Annalen der Physik, 2015, 527(7-8): 513-522. doi: 10.1002/andp.201500194 [39] Li J, Liu W, Li T, et al. Swimming microrobot optical nanoscopy [J]. Nano Letters, 2016, 16(10): 6604-6609. doi: 10.1021/acs.nanolett.6b03303 [40] Ashkin A. Optical trapping and manipulation of neutral particles using lasers [J]. Proc Natl Acad Sci, 1997, 94: 4853-4860. doi: 10.1073/pnas.94.10.4853 [41] Liu Xi, Hu Song, Tang Yan, et al. Selecting a proper microsphere to combine optical trapping with microsphere-assisted microscopy [J]. Applied Sciences, 2020, 10(9): 3127. doi: 10.3390/app10093127 [42] Liu X, Hu S, Tang Y. Coated high-refractive-index barium titanate glass microspheres for optically trapped microsphere super-resolution microscopy: a simulation study [J]. Photonics, 2020, 7(4): 84. doi: 10.3390/photonics7040084 [43] Wen Y, Yu H, Zhao W, et al. Scanning super-resolution imaging in enclosed environment by laser tweezer controlled superlens [J]. Biophysical Journal, 2020, 119(12): 2451-2460. doi: 10.1016/j.bpj.2020.10.032 [44] Huszka G, Yang H, Gijs M A M. Dielectric microsphere-based optical system for super-resolution microscopy[C]//2017 19th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS). IEEE, 2017: 2003-2006. [45] Huszka G, Gijs M A M. Custom adapter for extended field-of-view microsphere-based scanning super-resolution microscopy[C]//2018 IEEE Micro Electro Mechanical Systems (MEMS). IEEE, 2018: 700-703. [46] Huszka G, Krenger R, Gijs M A M. In vivo imaging with microsphere-based super-resolution microscopy[C]//2018 International Conference on Optical MEMS and Nanophotonics (OMN). IEEE, 2018: 1-2. [47] Chen L W, Y Zhou, Wu M X, et al. Remote-mode microsphere nano-imaging: new boundaries for optical microscopes [J]. Opto-Electronic Advances, 2018, 1(1): 4-10. doi: 10.29026/oea.2018.170001 [48] Yan B, Wang Z, Parker A L, et al. Superlensing microscope objective lens [J]. Applied Optics, 2017, 56(11): 3142-3147. doi: 10.1364/AO.56.003142 [49] Yan B, Song Y, Yang X, et al. Unibody microscope objective tipped with a microsphere: design, fabrication, and application in subwavelength imaging [J]. Applied Optics, 2020, 59(8): 2641-2648. doi: 10.1364/AO.386504 [50] Song Yang, Yang Xibin, Yan Bing, et al. Super-resolution imaging system based on integrated microsphere objective lens [J]. Acta Phys Sin, 2020, 69(13): 170-178. doi: 10.7498/aps.69.20191994 [51] Song Yang. Design of super-resolution imaging system based on integrated microsphere objective lens[D]. Shanghai: Shanghai University, 2020. (in Chinese) [52] Huszka G, Yang H, Gijs M A M. Microsphere-based super-resolution scanning optical microscope [J]. Optics Express, 2017, 25(13): 15079-15092. doi: 10.1364/OE.25.015079 [53] Huszka G, Gijs M A M. Turning a normal microscope into a super-resolution instrument using a scanning microlens array [J]. Scientific Reports, 2018, 8(1): 1-8. [54] Perrin S, Leong-Hoï A, Lecler S, et al. Microsphere-assisted phase-shifting profilometry [J]. Applied Optics, 2017, 56(25): 7249-7255. doi: 10.1364/AO.56.007249 [55] Wang F, Liu L, Yu P, et al. Three-dimensional super-resolution morphology by near-field assisted white-light interferometry [J]. Scientific Reports, 2016, 6: 24703. doi: 10.1038/srep24703 [56] Upputuri P K, Pramanik M. Microsphere-aided optical microscopy and its applications for super-resolution imaging [J]. Optics Communications, 2017, 404: 32-41. [57] Bezryadina A, Li J, Zhao J, et al. Localized plasmonic structured illumination microscopy with an optically trapped microlens [J]. Nanoscale, 2017, 9(39): 14907-14912. doi: 10.1039/C7NR03654J [58] Yang H, Moullan N, Auwerx J, et al. Superʜresolution biological microscopy using virtual imaging by a microsphere nanoscope [J]. Small, 2014, 10(9): 1712-1718. doi: 10.1002/smll.201302942 [59] Wen Y, Yu H, Zhao W, et al. Photonic nanojet sub-diffraction nano-fabrication with in situ super-resolution imaging [J]. IEEE Transactions on Nanotechnology, 2019, 18: 226-233. [60] Li Y, Liu X, Li B. Single-cell biomagnifier for optical nanoscopes and nanotweezers [J]. Light: Science & Applications, 2019, 8: 61.