Department of Chemical and Physical Sciences, University of Toronto, 3359 Mississauga Road, Mississauga, ON, L5L 1C6, Canada.
Photosynth Res. 2009 Nov-Dec;102(2-3):111-41. doi: 10.1007/s11120-009-9500-9. Epub 2009 Oct 23.
Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.
新兴的以及最常用的光学显微镜技术被回顾和在显微镜中的图像对比产生方法被提出，重点是非线性对比，如谐波产生和多光子激发荧光。非线性显微镜相对于线性显微镜技术具有许多优势，包括改进的深层组织成像、光学切片和活未染色样本的成像。尽管如此，除了多光子激发荧光之外，非线性显微镜还处于起步阶段，缺乏协议、用户和应用；因此，本综述重点介绍了非线性显微镜在研究光合生物中的潜力。呈现了非线性显微镜成像的例子，包括来自高等植物的分离的光收集天线复合物、淀粉颗粒、叶绿体、单细胞藻类 Chlamydomonas reinhardtii 以及蓝细菌 Leptolyngbya sp. 和 Anabaena sp. 在聚焦于非线性显微镜技术，二次和三次谐波产生和多光子激发荧光显微镜的同时，描述了其他新兴的非线性成像方式，并回顾了几种线性光学显微镜技术，以便清楚地描述它们的功能，并突出非线性显微镜的优势。
