Measuring incidence angle for through-the-objective total internal reflection fluorescence microscopy.
Measuring incidence angle for through-the-objective total internal reflection fluorescence microscopy.
复制标题
测量透过物镜全内反射荧光显微镜的入射角。
DOI：
10.1117/1.jbo.17.12.126007
复制
发表时间：
2012
影响因子：
3.5
通讯作者：
Burghardt,ThomasP
中科院分区：
文献类型：
--
作者：
Burghardt,ThomasP
全内反射荧光(TIRF)显微镜的激发光束从盖片和水样形成的玻璃/水界面的玻璃面入射到超过临界角度的位置。水侧瞬变场随距离界面的距离呈指数衰减，穿透深度随入射角的变化而变化。通过物镜的TIRF使激发的激光聚焦在背焦平面(BFP)，在物镜和盖板之间的小间隙产生接近界面的折射平行光束，这使得入射角的测量变得困难。目的轴向扫描不影响入射角，但平移由附着在界面水侧的荧光球的荧光质心检测到的光束与界面的交点。质心平移除以轴向平移是在目标轨迹上重复采样以获得最佳估计的入射角的切线。入射角是对聚焦光束在BFP上逐渐增大的径向位置进行测量的。通过物镜的TIRF显微镜，利用千分尺和中继镜将聚焦的光束定位在BFP上，校准入射角。校准取决于显微镜特性和TIRF物镜，适用于任何界面或样品。
Total internal reflection fluorescence (TIRF) microscopy has the exciting laser beam incident beyond critical angle from the glass side of a glass/aqueous interface formed by the coverslip and aqueous sample. The aqueous side evanescent field decays exponentially with distance from the interface with penetration depth depending on incidence angle. Through-the-objective TIRF has the exciting laser focused at the back focal plane (BFP) creating a refracted parallel beam approaching the interface in the small gap between objective and coverslip, making incidence angle challenging to measure. Objective axial scanning does not affect incidence angle but translates beam and interface intersection detected by the fluorescence center of mass from fluorescent spheres attached to the aqueous side of the interface. Center of mass translation divided by the axial translation is the tangent of the incidence angle that is sampled repeatedly over objective trajectory to obtain a best estimate. Incidence angle is measured for progressively larger radial positions of the focused beam on the BFP. A through-the-objective TIRF microscope, utilizing a micrometer and relay lenses to position the focused beam at the BFP, is calibrated for incidence angle. Calibration depends on microscope characteristics and TIRF objective and is applicable to any interface or sample.