Betz Timo, Teipel Jörn, Koch Daniel, Härtig Wolfgang, Guck Jochen, Käs Josef, Giessen Harald
Universität Leipzig, Fakultät für Physik und Geowissenschaften, Linnéstrasse 5, 04103 Leipzig, Germany.
J Biomed Opt. 2005 Sep-Oct;10(5):054009. doi: 10.1117/1.2114788.
Confocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300 nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination.
共聚焦显微镜和多光子显微镜是现代生命科学中的重要工具。它们能够对越来越多的荧光标记物进行快速且高分辨率成像，这些荧光标记物涵盖从荧光蛋白到量子点以及其他荧光团，用于分子在活细胞和生物体中的定位以及分子特性的定量检测。到目前为止，似乎只有一个物理限制是不可避免的。共聚焦显微镜和多光子显微镜都依赖激光作为激发源，并且它们的单色辐射仅允许有限数量的同时可用染料，这取决于所用显微镜中可用的特定激光谱线数量。我们通过在标准共聚焦显微镜中成功用锥形石英光纤产生的430至1300纳米的脉冲白光替代所有激发激光，克服了这一限制。通过这种易于重现的方法，展示了同时共聚焦显微镜和多光子显微镜。通过开发一种光谱宽度与汞灯相当的相干且强激光源，我们提供了激发任何所需荧光团组合的灵活性。
