Three‐photon excitation fluorescence imaging of biological specimens using an all‐solid‐state laser

D. Wokosin, V. Centonze, S. Crittenden, J. White
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引用次数: 80

Abstract

We demonstrate that three-photon excitation images of both fixed and living biological specimens can be readily obtained using an all-solid-state Nd:YLF laser excitation source. Optically sectioned images of fixed Caenorhabditis elegans embryos stained with DAPI and embryos triple-labeled with DAPI, fluorescein and Texas Red are presented. Time series images of a living LLC-PK cell stained with Hoechst 33342 during the progression from metaphase to telophase are also presented. The mode of excitation was inferred from the power-law of anthracene and Hoechst 33342 fluorescence versus incident laser power and an axial resolution comparison of anthracene fluorescence with two-photon excited Calcium Crimson fluorescence. Multiphoton excitation imaging is an attractive method for optically sectioning live specimens because of the lower levels of phototoxicity produced compared to other optical sectioning techniques. The combination of two- and three-photon excitation extends the capabilities of a multiple- photon imaging system since a single wavelength can provide localized excitation of a wide variety of fluorophores whose collective emission spectra can span the entire visible spectrum.
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用全固态激光器对生物标本进行三光子激发荧光成像
我们证明了使用全固态Nd:YLF激光激发源可以很容易地获得固定和活体生物标本的三光子激发图像。用DAPI染色固定秀丽隐杆线虫胚胎和用DAPI、荧光素和德克萨斯红三标记胚胎的光学切片图像。用Hoechst 33342染色的活的LLC-PK细胞从中期到末期的时间序列图像也被呈现。根据蒽和Hoechst 33342荧光与入射激光功率的幂律,以及蒽荧光与双光子激发的钙深红色荧光的轴向分辨率比较,推断了激发模式。由于与其他光学切片技术相比产生的光毒性较低,因此多光子激发成像是光学切片活体标本的一种有吸引力的方法。双光子和三光子激发的组合扩展了多光子成像系统的能力,因为单个波长可以提供各种荧光团的局部激发,其集体发射光谱可以跨越整个可见光谱。
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