Supporting Information for TIRF microscopy evanescent field calibration using tilted fluorescent microtubules.

Total internal reflection fluorescence microscopy has become a powerful tool to study the dynamics of sub-cellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field, that is, the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Here, we present a novel method to quantitatively characterize the illumination in total internal reflection fluorescence microscopy using tilted, fluorescently labelled, microtubules. We find that the evanescent field is well described by a single exponential function, with a penetration depth close to theoretically predicted values. The use of in vitro reconstituted microtubules as nanoscale probes results in a minimal perturbation of the evanescent field; excitation light scattering is eliminated and the refractive index of the sample environment is unchanged. The presented method has the potential to provide a generic tool for in situ calibration of the evanescent field.

全内反射荧光显微镜（Total internal reflection fluorescence microscopy）已成为研究底物表面附近亚细胞结构和单分子动力学的有力工具。然而，倏逝场（evanescent field）的穿透深度——即激发强度指数衰减至1/e时的距离——往往未被确定，这对成像结构的空间信息构成了限制。在此，我们提出一种新颖方法，利用倾斜的荧光标记微管（microtubules）定量表征全内反射荧光显微镜中的照明情况。研究发现，倏逝场可通过单指数函数很好地描述，其穿透深度与理论预测值接近。使用体外重构的微管作为纳米级探针，可使倏逝场的扰动降至最低；激发光散射被消除，且样品环境的折射率保持不变。该方法有望为倏逝场的原位校准提供一种通用工具。