Institute for Physical und Theoretical Chemistry, University of Braunschweig, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany.
J Fluoresc. 2009 Nov;19(6):1037-43. doi: 10.1007/s10895-009-0503-x. Epub 2009 Jul 10.
We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.
我们首次实现了双色双光子激光扫描显微镜（2c2pLSM），并利用该技术获取了细胞的紫外荧光图像。荧光是通过使用钛宝石飞秒激光器的基频和二次谐波的双色双光子吸收来产生的。800nm 光子和 400nm 光子的同时吸收与 266nm 的单光子吸收能量相当。这种激光扫描显微镜技术将钛宝石激发荧光显微镜的激发波长范围扩展到了紫外区。除了多光子显微镜的已知优势，如固有三维分辨率、减少光损伤和高穿透深度外，2c2pLSM 还为使用标准高数值孔径物镜进行紫外荧光成像提供了可能性。266nm 的有效激发波长特别适合色氨酸的激发光谱。因此，它是用于无标记荧光研究和固有蛋白质荧光成像的理想工具，这些荧光主要来自色氨酸。因此，可以使用非常灵敏的天然寿命探针来监测蛋白质反应或构象变化。对活 MIN-6 细胞的初步测量显示，细胞核和细胞质的紫外荧光寿命存在差异。通过监测生物素与亲和素的结合，进一步证明了这种方法的重要性。
