Department of Pathology and Cell Biology, Columbia University , New York, New York 10032, United States.
Section of Chemical and Materials Science, University of California, San Diego , La Jolla, California 92093, United States.
ACS Appl Mater Interfaces. 2017 Mar 8;9(9):7929-7940. doi: 10.1021/acsami.6b15322. Epub 2017 Feb 21.
The combination of near-infrared (NIR) and visible wavelengths in light microscopy for biological studies is increasingly common. For example, many fields of biology are developing the use of NIR for optogenetics, in which an NIR laser induces a change in gene expression and/or protein function. One major technical barrier in working with both NIR and visible light on an optical microscope is obtaining their precise coalignment at the imaging plane position. Photon upconverting particles (UCPs) can bridge this gap as they are excited by NIR light but emit in the visible range via an anti-Stokes luminescence mechanism. Here, two different UCPs have been identified, high-efficiency micro-UCPs and lower efficiency nano-UCPs, that respond to NIR light and emit visible light with high photostability even at very high NIR power densities (>25 000 Suns). Both of these UCPs can be rapidly and reversibly excited by visible and NIR light and emit light at visible wavelengths detectable with standard emission settings used for Green Fluorescent Protein (GFP), a commonly used genetically encoded fluorophore. However, the high efficiency micro-UCPs were suboptimal for NIR and visible light coalignment, due to their larger size and spatial broadening from particle-to-particle energy transfer consistent with a long-lived excited state and saturated power dependence. In contrast, the lower efficiency nano-UCPs were superior for precise coalignment of the NIR beam with the visible light path (∼2 μm versus ∼8 μm beam broadening, respectively) consistent with limited particle-to-particle energy transfer, superlinear power dependence for emission, and much smaller particle size. Furthermore, the nano-UCPs were superior to a traditional two-camera method for NIR and visible light path alignment in an in vivo Infrared-Laser-Evoked Gene Operator (IR-LEGO) optogenetics assay in the budding yeast Saccharomyces cerevisiae. In summary, nano-UCPs are powerful new tools for coaligning NIR and visible light paths on a light microscope.
近红外（NIR）和可见光在显微镜下的组合在生物学研究中越来越常见。例如，许多生物学领域正在开发使用近红外光进行光遗传学，其中近红外激光诱导基因表达和/或蛋白质功能的变化。在光学显微镜上同时使用近红外光和可见光的一个主要技术障碍是在成像平面位置获得它们的精确准直。上转换光子（UCP）可以弥合这一差距，因为它们被近红外光激发，但通过反斯托克斯发光机制在可见光范围内发射。在这里，已经确定了两种不同的 UCP，高效微 UCP 和低效率纳米 UCP，它们对近红外光有响应，并且即使在非常高的近红外功率密度（>25,000 太阳）下也具有高的光稳定性发出可见光。这两种 UCP 都可以被可见光和近红外光快速和可逆地激发，并在可见波长处发射光，这些光可以用 GFP（一种常用的遗传编码荧光蛋白）使用的标准发射设置检测到。然而，由于其较大的尺寸和粒子间能量转移引起的空间展宽，与长寿命激发态和饱和功率依赖性一致，高效微 UCP 不太适合近红外和可见光的准直。相比之下，低效率纳米 UCP 更适合近红外光束与可见光光路的精确准直（分别为2 μm 和8 μm 光束展宽），这与有限的粒子间能量转移、发射的超线性功率依赖性和更小的颗粒尺寸一致。此外，纳米 UCP 在芽殖酵母酿酒酵母的体内红外激光诱导基因操作（IR-LEGO）光遗传学测定中，在近红外和可见光光路对准方面优于传统的双相机方法。总之，纳米 UCP 是在显微镜下准直近红外和可见光光路的强大新工具。
