Wang Famin, Li Hangfeng, Ji Lin, Zhao Mengyuan, Miu Xin, Zhang Yunhai, Huang Wei, Wei Tongda
Appl Opt. 2021 Dec 10;60(35):10766-10771. doi: 10.1364/AO.433893.
A prominent challenge in single-molecule localization microscopy is the real-time, fast, and accurate localization of nano-objects moving in three-dimensional (3D) samples. A well-established method for 3D single-molecule localization is the double-helix pointspread-function (DH-PSF) engineering, which uses additional optical elements to make the PSF exhibit different rotation angles with different nanoparticle depths. However, the compact main lobe size, effective detection depth, and precise conversion between rotation angle and depth are necessary, posing challenges to the DH-PSF generation method. Here we generate a more compact DH-PSF using Fresnel-zone-based spiral phases, and the pure phase mask achieves high transmission efficiency. The final generated DH-PSFs have a linear rotation rate at each axial position, showing a more accurate rotation angle and depth conversion. The Cramer-Rao lower limit calculation results show that the axial depth of DH-PSF extends to ∼11µ with an axial localization precision of ∼45 at 3000 photons and average background noise of 15. We measured the diffusion coefficient of nanospheres in different concentrations of glycerol using the generated DH-PSF. The measured results are within 6% error from the theoretical values, indicating the superior performance of the DH-PSF for nanoparticle diffusion coefficient measurements.
单分子定位显微镜中的一个突出挑战是对在三维（3D）样品中移动的纳米物体进行实时、快速且准确的定位。一种成熟的3D单分子定位方法是双螺旋点扩展函数（DH-PSF）工程，它使用额外的光学元件使点扩展函数随纳米粒子深度不同呈现不同的旋转角度。然而，紧凑的主瓣尺寸、有效的检测深度以及旋转角度和深度之间的精确转换是必要的，这给DH-PSF生成方法带来了挑战。在此，我们使用基于菲涅尔区的螺旋相位生成了更紧凑的DH-PSF，并且纯相位掩膜实现了高传输效率。最终生成的DH-PSF在每个轴向位置具有线性旋转速率，显示出更精确的旋转角度和深度转换。克拉美-罗下限计算结果表明，在3000个光子和平均背景噪声为15的情况下，DH-PSF的轴向深度扩展到约11µ，轴向定位精度约为45。我们使用生成的DH-PSF测量了不同甘油浓度下纳米球的扩散系数。测量结果与理论值的误差在6%以内，表明DH-PSF在纳米粒子扩散系数测量方面具有卓越性能。
