Niederauer Christian, Blumhardt Philipp, Mücksch Jonas, Heymann Michael, Lambacher Armin, Schwille Petra
Opt Express. 2018 Aug 6;26(16):20492-20506. doi: 10.1364/OE.26.020492.
Total internal reflection fluorescence (TIRF) microscopy is a commonly used method for studying fluorescently labeled molecules in close proximity to a surface. Usually, the TIRF axial excitation profile is assumed to be single-exponential with a characteristic penetration depth, governed by the incident angle of the excitation laser beam towards the optical axis. However, in practice, the excitation profile does not only comprise the theoretically predicted single-exponential evanescent field, but also an additional non-evanescent contribution, supposedly caused by scattering within the optical path or optical aberrations. We developed a calibration slide to directly characterize the TIRF excitation field. Our slide features ten height steps ranging from 25 to 550 nanometers, fabricated from a polymer with a refractive index matching that of water. Fluorophores in aqueous solution above the polymer step layers sample the excitation profile at different heights. The obtained excitation profiles confirm the theoretically predicted exponential decay over increasing step heights as well as the presence of a non-evanescent contribution.
全内反射荧光（TIRF）显微镜是研究紧邻表面的荧光标记分子常用的方法。通常，TIRF轴向激发分布被假定为具有特征穿透深度的单指数形式，由激发激光束相对于光轴的入射角决定。然而，在实际中，激发分布不仅包含理论预测的单指数倏逝场，还包含一个额外的非倏逝贡献，推测是由光路中的散射或光学像差引起的。我们开发了一种校准载玻片来直接表征TIRF激发场。我们的载玻片有十个高度台阶，范围从25到550纳米，由折射率与水匹配的聚合物制成。聚合物台阶层上方水溶液中的荧光团在不同高度对激发分布进行采样。获得的激发分布证实了随着台阶高度增加理论预测的指数衰减以及非倏逝贡献的存在。
