Morgan C G, Mitchell A C
Biomedical Sciences Research Institute, School of Environmental and Life Sciences, University of Salford, Salford M5 4WT, UK.
J Microsc. 2006 Apr;222(Pt 1):48-57. doi: 10.1111/j.1365-2818.2006.01568.x.
Total internal reflection fluorescence microscopy is well known as a means of studying surface-bound structures in cell biology. It is usually measured either by coupling a light source to the sample using a prism or with a special objective where light passing through the periphery of the lens illuminates the contact region beyond the critical angle. In this study we present a new and simple approach to total internal reflection fluorescence microscopy where the sample is mounted on a cover slip prepared from a high-index upconverting glass-ceramic. Excitation of the cover slip with a low-cost near-infrared laser diode generates intense narrow-band visible emission within the cover slip, some of which is totally internally reflected. This emission gives rise to an evanescent wave at the interface and hence can excite surface-bound fluorescent species. Depending on the excitation conditions the cover slip can generate violet, green and red emission and hence can excite a wide range of fluorescent labels. Fluorescence emission from the sample can be detected in spectral regions where the direct emission from the cover slip is very weak. The advantages and limitations of the technique are discussed in comparison with conventional total internal reflection fluorescence microscopy measurements and prospects for novel total internal reflection fluorescence microscopy geometries are considered.
全内反射荧光显微镜是细胞生物学中研究表面结合结构的一种常用方法。通常通过使用棱镜将光源耦合到样品上，或者使用特殊物镜来进行测量，在特殊物镜中，穿过透镜周边的光以临界角之外的角度照射接触区域。在本研究中，我们提出了一种全新且简单的全内反射荧光显微镜方法，即将样品安装在由高折射率上转换玻璃陶瓷制备的盖玻片上。用低成本近红外激光二极管激发盖玻片，会在盖玻片内产生强烈的窄带可见光发射，其中一些光会发生全内反射。这种发射在界面处产生倏逝波，从而能够激发表面结合的荧光物质。根据激发条件，盖玻片可以产生紫色、绿色和红色发射，因此可以激发多种荧光标记。样品的荧光发射可以在盖玻片直接发射非常微弱的光谱区域中进行检测。与传统全内反射荧光显微镜测量相比，讨论了该技术的优缺点，并考虑了新型全内反射荧光显微镜几何结构的前景。
