Cecilie Vogt Klinik, Charité-University Medicine Berlin, Berlin, Germany.
Biophys J. 2010 Feb 17;98(4):715-23. doi: 10.1016/j.bpj.2009.10.035.
Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.
慢性炎症存在于各种器官中，如大脑，这意味着不同亚群的免疫细胞与靶器官的细胞相互作用。为了在形态和功能上监测这种细胞间通讯，能够在深部组织中可视化超过两种颜色是必不可少的。在这里，我们展示了基于光参量振荡器（OPO）的双光子激光扫描显微镜在中枢神经系统和免疫系统的难以接近的器官中进行动态活体成像的强大功能，对于需要使用荧光蛋白的长期病理机制研究（例如慢性神经炎症）具有特别重要的意义。将波长激发扩展到红外区域，克服了标准钛宝石激光激发的当前限制，从而实现了 1）具有很大不同激发和发射光谱的荧光团的同时成像（例如 GFP 衍生物和 RFP 衍生物）；以及 2）在更高分辨率下，在组织中的穿透深度更高（高达 80%），同时减少了光漂白和光毒性。该工具为深部组织成像开辟了新的机会，并将对生物科学和生物医学中活体应用的蛋白质荧光团的选择产生巨大影响，正如我们在这项工作中所展示的那样。
