3D-STORM超分辨成像中单分子轴向定位精度优化研究

Optimization of Single Molecules Axial Location Precision in 3D Stochastic Optical Reconstruction Microscopy

为定量描述柱透镜参数对三维成像过程中荧光分子轴向定位精度的影响,本文研究了柱透镜参数与点扩散函数椭圆率的相互关系.基于Olympus IX-83倒置荧光显微镜搭建成像系统,利用3个不同焦距柱透镜进行实验,通过柱透镜标定曲线的线性变化范围及该范围内曲线的斜率分析柱透镜参数选择上的优劣,优化并提高轴向定位的精度和深度.选择合适的柱透镜参数可实现标定曲线在焦平面上下1μm范围线性变化,轴向定位精度为16nm,并采用优化的标定曲线对肌动蛋白微丝进行三维超分辨成像.

The relationship between the ellipticity of point spread function and the focal length of cylindrical lens was investigated.Fluorescent inverted microscopey imaging system is based on Olympus IX-83.With three different focal length,the point spread function with custom built stochastic optical reconstruction microscopy instruments was measured.A method to evaluate the cylindrical lens was developed based on linear region and localization error.The results show that linear region of 1μm and axial localization error of 16 nm can be achieved with correct focal length.As the demonstration,three dimension super-resolution image of Actin filaments was reconstructed.