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双光子激发荧光(two-photon excited fluorescence, TPEF)显微是一种非线性光学显微技术, 具有高的时间分辨率和空间分辨率、高的信噪比和固有的三维层析分辨能力等优点. 传统的TPEF显微一般采用波长可调谐的超短脉冲激光器作为光源. 在实际应用中, 利用TPEF显微技术研究含有多种荧光团或未知成分的待测样品, 往往需要多次改变激发光的波长以获得对各种荧光团的最佳激发. 为了同时获取不同荧光团的荧光信号, 利用超连续谱激光光源实现了多色TPEF显微成像, 实验中无需调节波长, 能够同时获得具有两种不同发射波长的荧光标记的铃兰根茎切片样品的TPEF图像. 实验结果表明, 与传统的TPEF显微相比, 该方法能够同时获取含有多种荧光团的待测样品的高对比度TPEF图像, 具有系统结构简单、操作简便、信息量大等优点, 在生物医学和材料科学等领域具有广阔的应用前景.Two-photon excited fluorescence (TPEF) microscopy is a nonlinear optical microscopy technique. The advantages of TPEF microscopy include high temporal and spatial resolutions, high signal-to-noise ratio and inherent three-dimensional sectioning. In traditional TPEF microscopy, a wavelength tunable ultrashort pulsed laser is used as an excitation source. In practical applications, sample usually contains various fluorophores or unknown components. Therefore the excitation wavelength of the ultrafast laser has to be tuned to achieve optimal excitation efficiencies of various fluorophores. In order to acquire the fluorescent signals of different fluorophores simultaneously, we develop a multicolor TPEF microscope system based on a supercontinuum laser source. In experiments, TPEF images of Lily rhizome sample slide stained by two fluorescent dyes with different excitation and emission wavelengths are obtained without tuning the wavelength. Experimental results show that the high-contrast TPEF images of the sample with various fluorophores can be obtained simultaneously by using the multicolor TPEF microscope compared with by using traditional TPEF microscopy. The system is simple in structure, easy in operation, and can provide rich information about the sample, which allows it to be widely used in life and material sciences.
[1] Goeppert-Mayer M 1931 Ann. Phys. 401 273
[2] Kaiser W, Garrett C G B 1961 Phys. Rev. Lett. 7 229
[3] Denk W, Strickler J H, Webb W W 1990 Science 248 73
[4] Denk W 1994 Proc. Natl. Acad. Sci. 91 6629
[5] Strikler J H, Webb W W 1991 Opt. Lett. 16 1780
[6] Kawate Y, Ueki H, Hashimoto Y, Kawatav S 1995 Appl. Opt. 34 4105
[7] Krasieva T B, Stringari C, Liu F, Sun C H, Kong Y, Balu M, Meyskens F L, Gratton E, Tromberg B J 2013 J. Biom. Opt. 18 031107
[8] Tang Z L, Liang R S, Chang H S 2000 Acta Phys. Sin. 49 1080 (in Chinese) [唐志列, 梁瑞生, 常鸿森 2000 49 1080]
[9] Liu L X, Qu J L, Lin Z Y, Chen D N, Xu G X, Hu T, Guo B P, Niu H B 2006 Acta Phys. Sin. 55 6281 (in Chinese) [刘立新, 屈军乐, 林子扬, 陈丹妮, 许改霞, 胡涛, 郭宝平, 牛憨笨 2006 55 6281]
[10] Chen H W, Jin A J, Chen S P, Hou J, Lu Q S 2013 Chin. Phys. B 22 084205
[11] Liu S L, Chen D N, Liu W, Niu H B 2013 Acta Phys. Sin. 62 184210 (in Chinese) [刘双龙, 陈丹妮, 刘伟, 牛憨笨 2013 62 184210]
[12] Unruh J R, Price E S, Molla R G, Stehno-Bittel L, Johnson C K, Hui R 2006 Opt. Express 14 9825
[13] Li D, Zheng W, Qu J Y 2010 Proc. SPIE 7569 75692D
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[1] Goeppert-Mayer M 1931 Ann. Phys. 401 273
[2] Kaiser W, Garrett C G B 1961 Phys. Rev. Lett. 7 229
[3] Denk W, Strickler J H, Webb W W 1990 Science 248 73
[4] Denk W 1994 Proc. Natl. Acad. Sci. 91 6629
[5] Strikler J H, Webb W W 1991 Opt. Lett. 16 1780
[6] Kawate Y, Ueki H, Hashimoto Y, Kawatav S 1995 Appl. Opt. 34 4105
[7] Krasieva T B, Stringari C, Liu F, Sun C H, Kong Y, Balu M, Meyskens F L, Gratton E, Tromberg B J 2013 J. Biom. Opt. 18 031107
[8] Tang Z L, Liang R S, Chang H S 2000 Acta Phys. Sin. 49 1080 (in Chinese) [唐志列, 梁瑞生, 常鸿森 2000 49 1080]
[9] Liu L X, Qu J L, Lin Z Y, Chen D N, Xu G X, Hu T, Guo B P, Niu H B 2006 Acta Phys. Sin. 55 6281 (in Chinese) [刘立新, 屈军乐, 林子扬, 陈丹妮, 许改霞, 胡涛, 郭宝平, 牛憨笨 2006 55 6281]
[10] Chen H W, Jin A J, Chen S P, Hou J, Lu Q S 2013 Chin. Phys. B 22 084205
[11] Liu S L, Chen D N, Liu W, Niu H B 2013 Acta Phys. Sin. 62 184210 (in Chinese) [刘双龙, 陈丹妮, 刘伟, 牛憨笨 2013 62 184210]
[12] Unruh J R, Price E S, Molla R G, Stehno-Bittel L, Johnson C K, Hui R 2006 Opt. Express 14 9825
[13] Li D, Zheng W, Qu J Y 2010 Proc. SPIE 7569 75692D
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