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荧光显微成像技术具有标记能力强、信号强度高、实验成本低、成像过程简单且从活体到离体均可成像等特点, 在肿瘤细胞成像、药物分布体内探测等生物学分析成像研究中应用广泛, 但如何同时兼具宽视场和高分辨率是当前荧光显微成像领域的一大难点. 平面硅波导被发现可实现超薄样品大范围成像, 然而其需要溅射沉积或是离子束刻蚀等制备工艺, 相关工艺复杂且设备昂贵. 本文设计了一种基于皮秒激光直写的平面波导型荧光显微装置, 利用皮秒激光刻蚀玻璃表层快速制备微米级沟槽, 进一步通过旋涂SU-8光刻胶实现低成本、批量化制备玻璃基平面波导. 通过调整激光加工功率、频率、扫描速度等参数可以定制波导直径和深度. 采用罗丹明B荧光分子的显微探测实验, 验证了该激光直写玻璃基平面波导完全满足高分辨率和大视场的生物成像需要, 这种简易快速的加工手段能够有效提升荧光成像领域的经济效益和社会效益.Fluorescent microscopic imaging technology has the characteristics of strong labeling capability, high signal strength, low experimental cost, simple imaging process, and imaging from living to in vitro, which is widely used in biological analysis imaging research such as tumor cell imaging, drug distribution in vivo detection, but how to simultaneously have both a wide field of view and a high resolution is a major difficulty in the current field of fluorescence microscopic imaging. Planar silicon waveguides have been found to be able to achieve a wide range of imaging of ultra-thin samples. However, they require sputtering deposition or ion beam etching and other preparation processes. The related processes are complex and equipment required is expensive. In this work, a planar-waveguide-type fluorescence microscope device based on direct picosecond-laser-writing is designed, in which picosecond laser is used to etch the glass surface to rapidly prepare micron sized grooves, and the low-cost and batch-preparation of glass based planar waveguides is further realized by spinning SU-8 photoresist. The waveguide diameter and depth can be customized by adjusting laser processing power, frequency, scanning speed and other parameters. The microscopic detection experiment with using Rhodamine B fluorescent molecule verifies that the direct laser-writing glass based planar waveguide fully meets the requirements for biological imaging with high resolution and large field of view. This simple and rapid processing method can effectively improve the the fluorescence imaging.
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图 3 共聚焦表征 (a) 100 kHz; (b) 200 kHz; (c) 300 kHz; (d) 400 kHz; (e) 500 kHz 等重复频率下不同激光功率刻蚀的微沟槽深度; (f)刻蚀微沟槽显微镜图像
Fig. 3. Confocal characterization (a) 100 kHz; (b) 200 kHz; (c) 300 kHz; (d) 400 kHz; (e) 500 kHz, etc repetition frequencies, etching micro groove depth with different laser power; (f) Microscopic images of etched grooves.
图 4 (a)分束器显微镜图像; (b) 分束器通光表征
Fig. 4. (a) Beam splitter microscope image; (b) beam splitter clear characterization
图 5 (a)波导荧光探测示意图; (b)平面波导的荧光表征图和实物图; (c)输入路径局部放大图; (d)输出路径局部放大图
Fig. 5. (a) Schematic diagram of waveguide fluorescence detection; (b)fluorescence characterization and physical diagram of a planar waveguide; (c) input path local magnification; (d) output path local magnification.
图 6 (a)聚合物波导的集成制备图像; (b)蜿蜒型波导; (c)直线型波导
Fig. 6. (a) Integrated fabrication image of polymer waveguide; (b) Serpentine waveguide; (c) Linear waveguide.
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[1] Vysniauskas A, Lopez-Duarte I, Duchemin N, Vu T T, Wu Y L, Budynina E M, Volkova Y A, Cabrera E P, Ramirez-Ornelas D E, Kuimova M K 2017 Phys. Chem. Chem. Phys. 19 25252
Google Scholar
[2] Witte S, Negrean A, Lodder J C, de Kock C P J, Silva G T, Mansvelder H D, Groot M L 2011 Proc. Natl. Acad. Sci. U. S. A. 108 5970
Google Scholar
[3] Kuimova M K 2012 Phys. Chem. Chem. Phys. 14 12671
Google Scholar
[4] Yoon S, Kim M, Jang M, Choi Y, Choi W, Kang S, Choi W 2020 Nat. Rev. Phys. 2 141
Google Scholar
[5] Errico C, Pierre J, Pezet S, Desailly Y, Lenkei Z, Couture O, Tanter M 2015 Nature 527 499
Google Scholar
[6] Querard J, Zhang R K, Kelemen Z, Plamont M A, Xie X J, Chouket R, Roemgens I, Korepina Y, Albright S, Ipendey E, Volovitch M, Sladitschek H L, Neveu P, Gissot L, Gautier A, Faure J D, Croquette V, Le Saux T, Jullien L 2017 Nat. Commun. 8 2173
Google Scholar
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Google Scholar
[8] Schneckenburger H, Richter V 2021 Photonics 8 275
Google Scholar
[9] Moo E K, Abusara Z, Abu Osman N A, Pingguan-Murphy B, Herzog W 2013 J. Biomech. 46 2024
Google Scholar
[10] Nishiyama H, Suga M, Ogura T, Maruyama Y, Koizumi M, Mio K, Kitamura S, Sato C 2010 J. Struct. Biol. 169 438
Google Scholar
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Google Scholar
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Google Scholar
[13] 王美昌, 于斌, 张炜, 林丹樱, 屈军乐 2020 69 238701
Google Scholar
Wang M C, Yu B, Zhang W, Lin D Y, Qu J L 2020 Acta Phys. Sin. 69 238701
Google Scholar
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[15] Chatterjee K, Pratiwi F W, Wu F C M, Chen P L, Chen B C 2018 Appl. Spectrosc. 72 1137
Google Scholar
[16] Santi P A 2011 J. Histochem. Cytochem. 59 129
Google Scholar
[17] Maizel A, von Wangenheim D, Federici F, Haseloff J, Stelzer E H K 2011 Plant J. 68 377
Google Scholar
[18] Senarathna J, Yu H, Deng C, Zou A L, Issa J B, Hadjiabadi D H, Gil S, Wang Q H, Tyler B M, Thakor N V, Pathak A P 2019 Nat. Commun. 10 99
Google Scholar
[19] Sekiguchi K J, Shekhtmeyster P, Merten K, Arena A, Cook D, Hoffman E, Ngo A, Nimmerjahn A 2016 Nat. Commun. 7 11450
Google Scholar
[20] Zhou Y L, Li X 2017 Opt. Rev. 24 398
Google Scholar
[21] Glaser A K, Chen Y, Yin C B, Wei L P, Barner L A, Reder N P, Liu J T C 2018 Sci. Rep. 8 13878
Google Scholar
[22] Wu Y Q, Xu X, Wang J X, Zhang X, Shi G H 2021 Acta Opt. Sin. 41 2018001
Google Scholar
[23] 王海龙 2017 博士学位论文 (吉林: 吉林大学)
Wang H L 2017 Ph. D. Dissertation (Jilin: Jilin University) (in Chinese)
[24] O'Carroll D, Lieberwirth I, Redmond G 2007 Nat. Nanotechnol. 2 180
Google Scholar
[25] Lin Y, Gao C, Gritsenko D, Zhou R, Xu J 2018 Microfluid. Nanofluid. 22 97
Google Scholar
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Google Scholar
[27] Anders K E, Stefan B, Tung-Cheng W, Ralf H, Thomas Hr, Mark S 2021 ACS Photon. 8 1944
Google Scholar
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