为了提高在液态环境中对直径微米量级微球三维位置测量的空间分辨力，尤其是轴向的测量精度，将互相关算法对相似图像高精准的匹配特性与离焦成像法测量微球三维位置的思想相结合，讨论基于互相关匹配的离焦成像法测量微球三维位置的方法。实验表明，该方法对微球轴向位置4 min内连续定位的测量标准差约为0.64 nm，已实现对微球三维方向1 nm 阶跃变化的分辨测量，这在生物单分子动力学、粒子图像测速技术等研究领域具有重要意义。相同实验条件下，与同轴数字全息显微术对微球三维位置的对比测量和分析也初步说明了该方法在实际测量应用中的可行性与高精度的特点。

To improve the resolution of three-dimensional position measurement of micrometer scale spheres in liquid, especially the resolution in the axial axis, a method named three-dimensional microsphere tracking using off-focus images based on cross-correlation matching algorithm is developed. It is a combination of the cross-correlation algorithm which has high precision matching characteristics to similar images and the idea of tracking three-dimensional microspheres using off-focus images. As a result, the experiments show that a standard deviation of about 0.64 nm is reflected when tracking the same microsphere continuously in the axial direction in four minutes, and a resolution of 1 nm step measurement in all three axes is achieved. This is significant to the study of single biological molecule dynamics and particle image velocimetry etc. Under the same experimental conditions, the comparative measurement and analysis of three-dimensional microsphere tracking with digital in-line holographic microscopy show that the method exhibits high feasibility and resolution in practical measurement applications.