Institute of Physical Biology, University of South Bohemia, Zámek 136, 37333, Nové Hrady, Czech Republic.
Photosynth Res. 2009 Nov-Dec;102(2-3):169-75. doi: 10.1007/s11120-009-9498-z. Epub 2009 Sep 26.
Multi-color fluorescence emission from leaf tissues is presented as a powerful reporter on plant biochemistry and physiology that can be applied both at macro- and micro-scales. The blue-green fluorescence emission is typically excited by ultraviolet (UV) excitation. However, this approach cannot be applied in investigating intact leaf interior because the UV photons are largely absorbed in the epidermis of the leaf surface. This methodological barrier is eliminated by replacing the UV photon excitation by excitation with two infra-red photons of the same total energy. We demonstrate this approach by using two-photon excitation for microscopy of Arabidopsis thaliana leaves infected by pathogenic bacterium Pseudomonas syringae. The leaf structures are visualized by red chlorophyll fluorescence emission reconstructed in 3-D images while the bacteria are detected by the green emission of engineered fluorescence protein.
叶片组织的多色荧光发射是一种强大的植物生物化学和生理学报告器，可以在宏观和微观尺度上应用。蓝色-绿色荧光发射通常由紫外线 (UV) 激发。然而，这种方法不能应用于研究完整的叶片内部，因为 UV 光子在叶片表面的表皮中被大量吸收。通过用相同总能量的两个红外光子代替 UV 光子激发来消除这种方法学障碍。我们通过使用双光子激发显微镜观察感染了病原菌丁香假单胞菌的拟南芥叶片来证明这种方法。通过重建 3-D 图像中的红色叶绿素荧光发射来可视化叶片结构，而通过工程荧光蛋白的绿色发射来检测细菌。
