University of California San Diego, Department of Mechanical and Aerospace Engineering, La Jolla, CA 92093, USA.
Sci Rep. 2013;3:2133. doi: 10.1038/srep02133.
Total internal reflection fluorescence (TIRF) microscopy is a rapidly expanding optical technique with excellent surface sensitivity and limited background fluorescence. Commercially available TIRF systems are either objective based that employ expensive special high numerical aperture (NA) objectives or prism based that restrict integrating other modalities of investigation for structure-function analysis. Both techniques result in uneven illumination of the field of view and require training and experience in optics. Here we describe a novel, inexpensive, LED powered, waveguide based TIRF system that could be used as an add-on module to any standard fluorescence microscope even with low NA objectives. This system requires no alignment, illuminates the entire field evenly, and allows switching between epifluorescence/TIRF/bright field modes without adjustments or objective replacements. The simple design allows integration with other imaging systems, including atomic force microscopy (AFM), for probing complex biological systems at their native nanoscale regimes.
全内反射荧光（TIRF）显微镜是一种快速发展的光学技术，具有出色的表面灵敏度和有限的背景荧光。市售的 TIRF 系统要么基于物镜，采用昂贵的特殊高数值孔径（NA）物镜，要么基于棱镜，限制了整合其他结构功能分析研究模式的能力。这两种技术都会导致视场的照明不均匀，并且需要在光学方面进行培训和经验。在这里，我们描述了一种新颖、廉价、采用 LED 供电的基于波导的 TIRF 系统，即使使用低 NA 物镜，也可以作为任何标准荧光显微镜的附加模块使用。该系统不需要对准，均匀照亮整个视场，并允许在荧光显微镜/TIRF/明场模式之间切换，而无需进行调整或更换物镜。这种简单的设计允许与其他成像系统集成，包括原子力显微镜（AFM），用于在其天然纳米尺度范围内探测复杂的生物系统。
