基于激光干涉的结构光照明超分辨荧光显微镜系统
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摘要
结构光照明荧光显微术(SIM)是一种可突破阿贝衍射极限的宽场显微成像技术,因其非侵入、成像速度快及光损伤小等优点在生物医学研究中具有广泛的应用前景。从结构光照明显微成像系统基本原理出发,分析了超分辨图像重构算法原理、重构图像中伪影来源及优化方法;结合研制的线性/非线性结构光照明显微镜,详细讨论了基于激光干涉的SIM成像系统光机结构。重点讨论了系统的同步时序设计和光路中的几个关键技术问题。设计对比实验验证了自主开发的SIM重构算法的可靠性,并基于研制的线性SIM系统开展细胞骨架的成像实验。最后,对SIM技术在生物上的发展和应用提出展望。
Structured illumination fluorescence microscopy(SIM)is a wide field microscopy imaging technique which can circumvent the Abbe diffraction limit.It has various potential applications in the biomedical research due to its advantages of non-invasivity,fast imaging speed and low photon damage and so on.Based on the basic principle of structured illumination microscopy imaging system,the super-resolution image reconstruction algorithm,the sources of artifacts in reconstructed image and optimization methods are analyzed.The SIM imaging system based on laser interference is discussed combining custom built linear/non-linear structured illumination microscope.Design of the control system for hardware synchronization,and few key questions during optical alignment are discussed in detail.Imaging of cell skeleton is done with the linear SIM microscopy system and the comparative tests verify the reliability of the independent-developed image reconstruction algorithm.At last,the future improvement and application in biology of the SIM technique are discussed.
Structured illumination fluorescence microscopy(SIM)is a wide field microscopy imaging technique which can circumvent the Abbe diffraction limit.It has various potential applications in the biomedical research due to its advantages of non-invasivity,fast imaging speed and low photon damage and so on.Based on the basic principle of structured illumination microscopy imaging system,the super-resolution image reconstruction algorithm,the sources of artifacts in reconstructed image and optimization methods are analyzed.The SIM imaging system based on laser interference is discussed combining custom built linear/non-linear structured illumination microscope.Design of the control system for hardware synchronization,and few key questions during optical alignment are discussed in detail.Imaging of cell skeleton is done with the linear SIM microscopy system and the comparative tests verify the reliability of the independent-developed image reconstruction algorithm.At last,the future improvement and application in biology of the SIM technique are discussed.
引文
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[30]Lu Weimin.Optimization of structured illumination light field and characterization of system point spread function in structured illumination microscopy(SIM)[D].Changchun:Changchun Institute of Optics,Fine Mechanics and Physics,Chinese Academy of Sciences,2015.鹿伟民.SIM显微镜照明结构光场优化与系统点扩散函数表征[D].长春:中国科学院长春光学精密机械与物理研究所,2015.
[31]Heintzmann R.Structured illumination methods[M].New York:Springer,2010:265-279.
[32]Young L J,Str9hl F,Kaminski C F.A guide to structured illumination TIRF microscopy at high speed with multiple colors[J].Journal of Visualized Experiments,2016(111):53988.
[2]González S,Halpern A.Laser-scanning confocal microscopy[M].Heidelberg:Springer,2007:39-46.
[3]Rayleigh L.Investigations in optics,with special reference to the spectroscope[J].Philosophical Magazine,1879,8(49):261-274.
[4]Huang B,Wang W,Bates M,et al.Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy[J].Science,2008,319(5864):810-813.
[5]Rust M J,Bates M,Zhuang X.Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy(STORM)[J].Nature Methods,2006,3(10):793-795.
[6]Betzig E,Patterson G H,Sougrat H,et al.Imaging intracellular fluorescent proteins at nanometer resolution[J].Science,2006,313(5793):1642-1645.
[7]Hess S T,Girirajan T P K,Mason M D.Ultra-high resolution imaging by fluorescence photoactivation localization microscopy[J].Biophysical Journal,2006,91(11):4258-4272.
[8]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.
[9]Klar T A,Jakobs S,Dyba M,et al.Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(15):8206-8210.
[10]Heintzmann R,Cremer C G.Laterally modulated excitation microscopy:improvement of resolution by using a diffraction grating[C].Proceedings of the SPIE,1999,3568:185-196.
[11]Gustafsson M G.Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy[J].Journal of Microscopy,2000,198(2):82-87.
[12]Heintzmann R,Jovin T M,Cremer C.Saturated patterned excitation microscopy-a concept for optical resolution improvement[J].Journal of the Optical Society of America A:Optics Image Science&Vision,2002,19(8):1599-1609.
[13]Gustafsson M G L.Nonlinear structured-illumination microscopy:Wide-field fluorescence imaging with theoretically unlimited resolution[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(37):13081-13086.
[14]Hirvonen L,Mandula O,Wicker K,etal.Structured illumination microscopy using photoswitchable fluorescent proteins[C].Proceedings of the SPIE,2008,6861:68610L.
[15]Rego E H,Lin S,Macklin J J,et al.Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50nm resolution[J].Proceedings of the National Academy of Sciences of the United States of America,2011,109(3):E135-E143.
[16]Gustafsson M G,Shao L,Carlton P M,et al.Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination[J].Biophysical Journal,2008,94(12):4957-4970.
[17]Hirvonen L M,Wicker K,Mandula O,et al.Structured illumination microscopy of a living cell.[J].European Biophysics Journal 2009,38(6):807-812.
[18]Kner P,Chhun B B,Griffis E R,et al.Super-resolution video microscopy of live cells by structured illumination[J].Nature Methods,2009,6(5):339-342.
[19]Shao L,Kner P,Rego E H,et al.Super-resolution 3Dmicroscopy of live whole cells using structured illumination[J].Nature Methods,2011,8(12):1044-1046.
[20]Fiolka R,Shao L,Rego E H,et al.Time-lapse two-color 3Dimaging of live cells with doubled resolution using structured illumination[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(14):5311-5315.
[21]Dan D,Lei M,Yao B,et al.DMD-based LED-illumination super-resolution and optical sectioning microscopy[J].Scientific Reports,2013,3:1116.
[22]Li D,Shao L,Chen B C,et al.Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics[J].Science,2015,349(6251):527.
[23]Heintzmann R,Benedetti P A.High-resolution image reconstruction in fluorescence microscopy with patterned excitation[J].Applied Optics,2006,45(20):5037-5045.
[24]Kai Wicker.Non-iterative determination of pattern phase in structured illumination microscopy using auto-correlations in Fourier space[J].Optics Express,2013,21(21):24692-24701.
[25]Müller M,M9nkem9ller V,Hennig S,et al.Open-source image reconstruction of super-resolution structured illumination microscopy data in image[J].Nature Communications,2016,7:10980.
[26]Kai W.Increasing resolution and light efficiency in fluorescence microscopy[D].London:King′s College,2010.
[27]Shroff S A,Fienup J R,Williams D R.Phase-shift estimation in sinusoidally illuminated images for lateral superresolution[J].Journal of the Optical Society of America A:Optics Image Science&Vision,2009,26(2):413-424.
[28]Shroff S A,Fienup J R,Williams D R.Lateral superresolution using aposteriori phase shift estimation for a moving object:experimental results[J].Journal of the Optical Society of America A:Optics Image Science&Vision,2010,27(8):1770-1782.
[29]Chu K,Mcmillan P J,Smith Z J,et al.Image reconstruction for structured-illumination microscopy with low signal level[J].Optics Express,2014,22(7):8687-8702.
[30]Lu Weimin.Optimization of structured illumination light field and characterization of system point spread function in structured illumination microscopy(SIM)[D].Changchun:Changchun Institute of Optics,Fine Mechanics and Physics,Chinese Academy of Sciences,2015.鹿伟民.SIM显微镜照明结构光场优化与系统点扩散函数表征[D].长春:中国科学院长春光学精密机械与物理研究所,2015.
[31]Heintzmann R.Structured illumination methods[M].New York:Springer,2010:265-279.
[32]Young L J,Str9hl F,Kaminski C F.A guide to structured illumination TIRF microscopy at high speed with multiple colors[J].Journal of Visualized Experiments,2016(111):53988.