留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

长时程双光子成像技术

徐依雯 张运海 杨皓旻 刘创 唐玉国

徐依雯, 张运海, 杨皓旻, 刘创, 唐玉国. 长时程双光子成像技术[J]. 中国光学, 2018, 11(3): 337-343. doi: 10.3788/CO.20181103.0337
引用本文: 徐依雯, 张运海, 杨皓旻, 刘创, 唐玉国. 长时程双光子成像技术[J]. 中国光学, 2018, 11(3): 337-343. doi: 10.3788/CO.20181103.0337
XU Yi-wen, ZHANG Yun-hai, YANG Hao-min, LIU Chuang, TANG Yu-guo. Long-time two-photon imaging technology[J]. Chinese Optics, 2018, 11(3): 337-343. doi: 10.3788/CO.20181103.0337
Citation: XU Yi-wen, ZHANG Yun-hai, YANG Hao-min, LIU Chuang, TANG Yu-guo. Long-time two-photon imaging technology[J]. Chinese Optics, 2018, 11(3): 337-343. doi: 10.3788/CO.20181103.0337

长时程双光子成像技术

doi: 10.3788/CO.20181103.0337
基金项目: 

国家重点研发计划 No.2017YFC0110303

国家重大科研装备研制项目 No.ZDYZ2013-1

江苏省基础研究计划青年基金项目 No.BK20160363

苏州应用基础研究计划项目 No.SYG201510

详细信息
    作者简介:

    徐依雯(1993—), 女, 江苏泰州人,硕士研究生,2015年于西安交通大学获得学士学位, 现为中国科学院苏州生物医学工程技术研究所硕士研究生,主要从事共聚焦激光扫描荧光显微成像方面的研究。E-mail:xuyiwen15@mails.ucas.edu.cn

    张运海(1975—),男,湖北襄阳人,博士,研究员,1998年、2006年于南京航空航天大学分别获得学士、博士学位,主要从事共聚焦显微成像技术、超分辨受激辐射损耗(STED)成像技术等方面的研究。E-mail:zhangyh@sibet.ac.cn

  • 中图分类号: O439

Long-time two-photon imaging technology

Funds: 

National Key R & D Program No.2017YFC0110303

National Major Scientific Research and Equipment Development Project No.ZDYZ2013-1

Youth Foundation of Jiangsu Province Basic Research Program No.BK20160363

Suzhou Applied Basic Research Project No.SYG201510

More Information
  • 摘要: 双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。
  • 图  1  物方像素点扫描示意图

    Figure  1.  Spot scanning diagram of objective pixel

    图  2  OL-TP像素光照时间优化过程示意图

    Figure  2.  Diagram of optimized process for pixel lighting time in OL-TP

    图  3  像素值与像素光照时间关系示意图

    Figure  3.  Relationship between pixel value and lighting time

    图  4  (a) 传统双光子图像;(b)像素光照时间分布图;(c)实际像素值分布图;(d)OL-TP重建图像

    Figure  4.  (a)Conventional two-photon image; (b)distribution of pixel lighting time; (c)distribution of real pixel value; (d)reconstructed image of OL-TP

    图  5  OL-TP成像系统结构示意图

    Figure  5.  Diagram of OL-TP image system

    图  6  (a) 传统双光子图像。(b)OL-TP重建图像

    Figure  6.  (a)Conventional two-photon image. (b)Reconstructed image of OL-TP

    图  7  (a) 传统双光子。(b)OL-TP重建图像

    Figure  7.  (a)Conventional two-photon image. (b)Reconstructed image of OL-TP

  • [1] CRISAFI F, KUMAR V, PERRI A, et al.. Multimodal nonlinear microscope based on a compact fiber-format laser source[J]. Spectrochimica Acta Part A Molecular & Biomolecular Spectroscopy, 2017, 188:135-140. http://cn.bing.com/academic/profile?id=ef8ab5e58d2ad5ba69a3d119d127365c&encoded=0&v=paper_preview&mkt=zh-cn
    [2] DENK W, STRICKLER J H, WEBB W W. Two-photon laser scanning fluorescence microscopy[J]. Science, 1990, 248(4951):73-76. doi: 10.1126/science.2321027
    [3] 李茜. 飞秒激光双光子荧光生物显微成像研究[D]. 哈尔滨: 哈尔滨工业大学, 2014. http://cdmd.cnki.com.cn/Article/CDMD-10213-1014083095.htm

    LI Q. Femtosecond laser two-photon fluorescence biological microscopy[D]. Harbin: Harbin Institute of Technology, 2014. (in Chinese) http://cdmd.cnki.com.cn/Article/CDMD-10213-1014083095.htm
    [4] 李奇峰, 沙乾坤, 王洋, 等.基于非线性光学的2PE-STED显微技术的发展与应用[J].纳米技术与精密工程, 2015(2):81-89. http://www.cnki.com.cn/Article/CJFDTotal-NMJM201502001.htm

    LI Q F, SHA Q K, WANG Y, et al.. Development and application of 2PE-STED microscopy based on nonlinear optics[J]. Nanotechnology and Prescision Engineering, 2015(2):81-89.(in Chinese) http://www.cnki.com.cn/Article/CJFDTotal-NMJM201502001.htm
    [5] MILLER D R, JARRETT J W, HASSAN A M, et al.. Deep tissue imaging with multiphoton fluorescence microscopy[J]. Current Opinion in Biomedical Engineering, 2017, 4:32-39. doi: 10.1016/j.cobme.2017.09.004
    [6] TSAKANOVA G, ARAKELOVA E, AYVAZYAN V, et al.. Two-photon microscopy imaging of oxidative stress in human living erythrocytes[J]. Biomedical Optics Express, 2017, 8(12):5834-5846. doi: 10.1364/BOE.8.005834
    [7] 熊大曦, 刘云, 梁永, 等.共振扫描显微成像中的图像畸变校正[J].光学精密工程, 2015, 23(10):2971-2979. http://www.eope.net/gxjmgc/article/2015/2015-10-2971.htm

    XIONG D X, LIU Y, LIANG Y, et al.. Correction of distortion in microscopic imaging with resonant scanning[J]. Opt. Precision Eng., 2015, 23(10):2971-2979.(in Chinese) http://www.eope.net/gxjmgc/article/2015/2015-10-2971.htm
    [8] 马军, 田玉顺.双光子荧光影像技术在药物研发中的应用前景[J].中国现代应用药学, 2016, 33(3):381-385. http://www.cqvip.com/QK/95393A/201603/668383114.html

    MA J, TIAN Y SH. Application prospect of two-photon fluorescence imaging technology in drug development[J]. Chinese Journal of Modern Applied Pharmacy, 2016, 33(3):381-385.(in Chinese) http://www.cqvip.com/QK/95393A/201603/668383114.html
    [9] 崔权, 陈忠云, 张智红, 等.多色双光子成像技术进展[J].激光与光电子学进展, 2017(6):10-23. http://www.opticsjournal.net/ViewFullPDF.htm?aid=OJ170608000009LiOkRn

    CUI Q, CHEN ZH Y, ZHANG ZH H, et al.. Recent advances in multicolor two-photon imaging technique[J]. Laser & Optoelectronics Progress, 2017, 54(6):060002.(in Chinese) http://www.opticsjournal.net/ViewFullPDF.htm?aid=OJ170608000009LiOkRn
    [10] LEFORT C. A review of biomedical multiphoton microscopy and its laser sources[J]. Journal of Physics D Applied Physics, 2017, 50(42):423001. doi: 10.1088/1361-6463/aa8050
    [11] TISCHBIREK C H, BIRKNER A, KONNERTH A. In vivo deep two-photon imaging of neural circuits with the fluorescent Ca2+ indicator Cal-590[J]. Journal of Physiology, 2017, 595(10):3097-3105. doi: 10.1113/JP272790
    [12] 夏伟强, 周源, 石明.双光子显微成像技术的新进展[J].中国医疗器械杂志, 2011, 35(3):204-208. http://www.docin.com/p-304442607.html

    XIA W Q, ZHOU Y, SHI M. Advances in two-photon imaging technology[J]. Chinese Journal of Medical Instrumentation, 2011, 35(3):204-208.(in Chinese) http://www.docin.com/p-304442607.html
    [13] HOPT A, NEHER E. Highly nonlinear photodamage in two-photon fluorescence microscopy[J]. Biophys J, 2001, 80(4):2029-2036. doi: 10.1016/S0006-3495(01)76173-5
    [14] STEPHENS D J, ALLAN V J. Light microscopy techniques for live cell imaging[J]. Science, 2003, 300(5616):82-86. doi: 10.1126/science.1082160
    [15] STAUDT T, ENGLER A, RITTWEGER E, et al.. Far-field optical nanoscopy with reduced number of state transition cycles[J]. Optics Express, 2011, 19(6):5644-5657. doi: 10.1364/OE.19.005644
    [16] HOEBE R A, VAN OVEN C H, GADELLA T W J, et al.. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging[J]. Nat. Biotech., 2007, 25(2):249-253. doi: 10.1038/nbt1278
    [17] MONDAL P P, DIASPRO A. Reduction of higher-order photobleaching in two-photon excitation microscopy[J]. Physical Review E Statistical Nonlinear & Soft Matter Physics, 2007, 75(6 Pt 1):061904. http://cn.bing.com/academic/profile?id=f241922887607447309f38575d1a6513&encoded=0&v=paper_preview&mkt=zh-cn
    [18] PATTERSON, GEORGE H, DAVID W. Photobleaching in two-photon excitation microscopy[J]. Biophysical Journal, 2000, 78(4):2159-2162. doi: 10.1016/S0006-3495(00)76762-2
    [19] 张运海, 杨皓旻, 孔晨晖.激光扫描共聚焦光谱成像系统[J].光学精密工程, 2014, 22(6):1446-1453. http://www.eope.net/gxjmgc/CN/abstract/abstract15267.shtml

    ZHANG Y H, YANG H M, KONG CH H. Spectral imaging system on laser scanning confocal microscopy[J]. Opt. Precision Eng., 2014, 22(6):1446-1453.(in Chinese) http://www.eope.net/gxjmgc/CN/abstract/abstract15267.shtml
    [20] EGGELING C, WILLIG K I, SAHL S J, et al.. Lens-based fluorescence nanoscopy[J]. Quarterly Reviews of Biophysics, 2015, 48(2):178-243. doi: 10.1017/S0033583514000146
    [21] 张达奇. 全参考图像主观质量客观评价算法研究[D]. 苏州: 苏州大学, 2014. http://cdmd.cnki.com.cn/Article/CDMD-10285-1014298659.htm

    ZHANG D Q. Research on objective algorithms for full-reference image quality assessment[D]. Soochow: Soochow University, 2014. (in Chinese) http://cdmd.cnki.com.cn/Article/CDMD-10285-1014298659.htm
    [22] 褚江, 陈强, 杨曦晨.全参考图像质量评价综述[J].计算机应用研究, 2014, 31(1):13-22. http://www.cnki.com.cn/Article/CJFDTotal-JSYJ201401004.htm

    CHU J, CHEN Q, YANG X CH. Review on full reference image quality assessment algorithms[J]. Application Research of Computers, 2014, 31(1):13-22.(in Chinese) http://www.cnki.com.cn/Article/CJFDTotal-JSYJ201401004.htm
  • 加载中
图(7)
计量
  • 文章访问数:  639
  • HTML全文浏览量:  221
  • PDF下载量:  177
  • 被引次数: 0
出版历程
  • 收稿日期:  2018-01-23
  • 修回日期:  2018-02-13
  • 刊出日期:  2018-06-01

目录

    /

    返回文章
    返回