论文标题
解决单个发射极定位显微镜中轴向距离测量中的系统错误
Addressing systematic errors in axial distance measurements in single-emitter localization microscopy
论文作者
论文摘要
点发射器的纳米级定位对于光学荧光显微镜的几种方法至关重要，包括单分子超分辨率成像和跟踪。虽然本地化程序的精度一直是广泛研究的主题，但本地化准确性的强调较少，部分原因是在相关几何学中生产了一个已知的三维位置的实验样本的挑战。我们报告了一个新的实验系统，该系统在高数量孔径定位显微镜中繁殖了广泛的几何形状，其中分子位于以油浸泡物镜成像的玻璃盖上方的水培养基中。我们展示了一个校准程序，该程序可以测量依赖深度依赖的点扩散函数（PSF），以进行开放孔径成像以及具有索引不匹配的工程PSF成像。我们揭示了该系统中焦平面位置的复杂，深度变化的行为，并讨论了这种行为的共同近似值产生的轴向定位偏见。我们将结果与理论计算进行比较。
Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.
