Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence
Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence
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DOI：
10.1117/1.2161018
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发表时间：
2006-01-01
影响因子：
3.5
通讯作者：
Axelrod, D
中科院分区：
文献类型：
--
作者：
Mattheyses, AL;Axelrod, D
全内反射荧光 (TIRF) 显微镜产生一个薄的激发场（倏逝场），名义上呈指数衰减。该场非常适合选择性激发盖玻片/样品界面附近的荧光团。我们提出了一种实验方法，可以通过在折射率匹配溶液中对低折射率荧光标记球形珠进行显微观察来直接测量渐逝场的深度和轴向轮廓。为了演示该技术，通过激光激发设置了透过物镜 TIRF。在此配置中，由 1.45 数值孔径 (NA) 或 1.65 NA 物镜创建的渐逝场的轴向轮廓非常适合双指数。在盖玻片/样品界面处，约 90% 的倏逝场由指数表示，其衰减率与理论倏逝场的预期一致；剩余 10% 的场由具有更长衰减常数的指数表示，并被识别为散射。这里提出的方法对于研究基于激光和基于汞弧的直通物镜 TIRF 系统中渐逝场的质量和轴向轮廓特别有用，在这些系统中，照明光学器件中可能会发生大量的光散射。 (c) 2006 年光电仪器工程师协会。
Total internal reflection fluorescence (TIRF) microscopy produces a thin excitation field (the evanescent field) that nominally decays exponentially. This field is ideal for selective excitation of fluorophores near the coverslip/ sample interface. We present an experimental method, where the depth and axial profile of the evanescent field can be measured directly by microscopic observation of low refractive index fluorescently labeled spherical beads in an index-matched solution. To demonstrate the technique, through-the-objective TIRF is set up with laser excitation. In this configuration, the axial profile of the evanescent field created by either a 1.45-numerical aperture (NA) or a 1.65-NA objective fits well to a double exponential. At the coverslip/ sample interface, about 90% of the evanescent field is represented by an exponential with a decay rate consistent with that expected for a theoretical evanescent field; the remaining 10% of the field is represented by an exponential with a much longer decay constant and is identified as scattering. The approach presented here is particularly useful for investigating the quality and axial profile of the evanescent field in both laser-based and mercury arc-based through-the-objective TIRF systems where a significant amount of light scattering can occur in the illumination optics. (c) 2006 Society of Photo-Optical Instrumentation Engineers.