Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy
Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy
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DOI：
10.1073/pnas.93.20.10763
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发表时间：
1996-10-01
影响因子：
11.1
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Xu, C;Zipfel, W;Webb, WW
非线性激光扫描荧光显微镜实现了对生物制剂中动态结构和生化过程的内在三维分辨显微成像。由于非线性显微镜作为一种强大的生物物理仪器的出现，对有用的双光子和三光子激发光谱的研究现在已经发现了一种虚拟的艺术家调色板，包括化学指示剂、荧光标记和天然生物荧光团，包括NADH、黄素和绿色荧光蛋白，它们适用于活的生物制剂，紫外和可见吸收分子的超过25个双光子激发光谱揭示了有用的横截面，对于近红外吸收，一些方便地蓝移，三光子荧光团激发光谱的测量现在定义了相对良性波长的替代窗口，以激发更深的紫外荧光团。非线性激发的固有光学切片能力为成像提供了三维分辨率，并避免了失焦背景和光损伤。在这里，测量的非线性激发光谱和它们的光物理特性，使非线性激光显微镜生物成像进行了描述。
Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes In living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations, More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption, Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.