Mattheyses Alexa L, Axelrod Daniel
University of Michigan, Biophysics Research Division, Ann Arbor, Michigan 48109, USA.
J Biomed Opt. 2006 Jan-Feb;11(1):014006. doi: 10.1117/1.2161018.
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.
全内反射荧光（TIRF）显微镜产生一个薄的激发场（倏逝场），该场理论上呈指数衰减。这个场非常适合在盖玻片/样品界面附近选择性激发荧光团。我们提出了一种实验方法，通过在折射率匹配的溶液中对低折射率荧光标记的球形珠子进行显微镜观察，可以直接测量倏逝场的深度和轴向轮廓。为了演示该技术，使用激光激发设置了透射物镜TIRF。在这种配置下，由数值孔径为1.45或1.65的物镜产生的倏逝场的轴向轮廓与双指数函数拟合得很好。在盖玻片/样品界面处，约90%的倏逝场由一个指数表示，其衰减率与理论倏逝场预期的衰减率一致；其余10%的场由一个衰减常数长得多的指数表示，被确定为散射。这里提出的方法对于研究基于激光和基于汞弧的透射物镜TIRF系统中倏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。
