We present a three-dimensional (3D) imaging technique for the fast tracking of microscopic objects in a fluid environment. Our technique couples digital holographic microscopy with three-dimensional localization via parabolic masking. Compared with existing approaches, our method reconstructs 3D volumes from single-plane images, which greatly simplifies image acquisition, reduces the demand on microscope hardware, and facilitates tracking higher densities of microscopic particles while maintaining similar levels of precision. We demonstrate utility of this method in magnetic tweezer experiments, opening their use to multiplexed single-molecule force spectroscopy assays, which were previously limited by particle crowding and fast dissociation times. We propose that our technique will also be useful in other applications that involve the tracking of microscopic objects in three dimensions, such as studies of microorganism motility and 3D flow characterization of microfluidic devices.

我们提出了一种三维（3D）成像技术，用于快速跟踪流体环境中的微观物体。我们的技术通过抛物线掩蔽将数字全息显微镜与三维定位结合起来。与现有方法相比，我们的方法从单平面图像重建 3D 体积，这大大简化了图像采集，减少了对显微镜硬件的需求，并有助于跟踪更高密度的微观粒子，同时保持相似的精度水平。我们在磁镊实验中展示了这种方法的实用性，将其用于多重单分子力谱分析，该分析以前受到粒子拥挤和快速解离时间的限制。