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Theoretical background of light-emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane-associated proteins-Part II.
Journal of Biophotonics ( IF 2.8 ) Pub Date : 2020-02-05 , DOI: 10.1002/jbio.201960181
Michael Schaefer 1 , Hermann Kalwa 1
Affiliation  

The selective microscopic imaging of the plasma membrane and adjacent structures by total internal reflection fluorescence (TIRF) microscopy is a versatile and frequently used technique in cell biology. A reduction of imaging artifacts in objective‐type TIRF microscopy can be achieved by circular or multi‐spot laser illumination or by using noncoherent light sources that are projected into the back focal plane as a light annulus. Light‐emitting diode (LED)‐based TIRF excitation is a recent advancement of the latter strategy. While some basic principles of LED‐TIRF remain the same as in laser‐based methods, the calculation of penetration depth, the flatness of illumination and the amount of available illumination power differ. This study provides the theoretical framework for the construction and adjustment of LED‐TIRF. Using state‐of‐the art high power LED emitters, LED‐TIRF achieves excitation efficiencies that are comparable to laser‐based systems and homogenously illuminate the entire field of view, thus, allowing variation of the penetration depth or quantitative photobleaching‐assisted imaging protocols. Using autofluorescent transmembrane, soluble and membrane‐attached fusion proteins, we provide examples for a photobleaching‐based assessment of the exchange kinetics of proteins within living human endothelial cells.image

中文翻译:

膜相关蛋白的发光二极管全内反射荧光显微镜的理论背景和光漂白寿命分析-第二部分。

通过全内反射荧光(TIRF)显微镜对质膜和邻近结构进行选择性显微成像是细胞生物学中一种通用且常用的技术。可以通过圆形或多点激光照明或通过使用非相干光源(作为光环投射到后焦平面中)来减少物镜TIRF显微镜中的成像伪影。基于发光二极管(LED)的TIRF激励是后一种策略的最新进展。尽管LED‐TIRF的一些基本原理与基于激光的方法相同,但穿透深度,照明的平坦度和可用照明功率的计算却有所不同。本研究为LED-TIRF的构建和调整提供了理论框架。使用最先进的大功率LED发射器,LED-TIRF可获得与基于激光的系统相当的激发效率,并均匀照明整个视场,从而允许改变穿透深度或定量的光漂白辅助成像方案。我们使用自发荧光的跨膜,可溶性和膜附着融合蛋白,为基于光漂白的人内皮细胞内蛋白交换动力学评估提供了实例。图片
更新日期:2020-02-05
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