We report a fast autofocusing and accurate 3D tracking scheme for a digital hologram (DH) that intrinsically exploits a polarization microscope setup with two off-axis illumination beams having different polarization. This configuration forms twin-object images that are recorded in a digital hologram by angular and polarization multiplexing technique. We show that the separation of the two images on the recording plane follows a linear relationship with the defocus distance and indicates the defocus direction. Thus, in the entire field of view (FOV), the best focus distance of each object can be directly retrieved by identifying the respective separation distance with a cross-correlation algorithm, at the same time, 3D tracking can be performed by calculating the transverse coordinates of the two images. Moreover, we estimate this linear relationship by utilizing the numerical propagation calculation based on a single hologram, in which the focus distance of one of the objects in the FOV is known. We proved the proposed approach in accurate 3D tracking through multiple completely different experimental cases, i.e., recovering the swimming path of a marine alga (tetraselmis) in water and fast refocusing of ovarian cancer cells under micro-vibration stimulation. The reported experimental results validate the proposed strategy’s effectiveness in dynamic measurement and 3D tracking without multiple diffraction calculations and any precise knowledge about the setup. We claim that it is the first time that a holographic polarization multiplexing setup is exploited intrinsically for 3D tracking and/or fast and accurate refocusing. This means that almost any polarization DH setup, thanks to our results, can guarantee accurate focusing along the optical axis in addition to polarization analysis of the sample, thus overcoming the limitation of the poor axial resolution.

中文翻译：

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利用全息偏振显微镜进行快速自动对焦和 3D 跟踪


我们报告了一种数字全息图 (DH) 的快速自动对焦和精确 3D 跟踪方案，该方案本质上利用了具有两个具有不同偏振的离轴照明光束的偏振显微镜设置。这种配置形成双物体图像，通过角度和偏振复用技术记录在数字全息图中。我们表明，记录平面上两个图像的分离与离焦距离呈线性关系，并指示离焦方向。因此，在整个视场（FOV）内，通过互相关算法识别各自的间隔距离，可以直接检索每个物体的最佳焦距，同时可以通过计算横向距离来进行3D跟踪。两个图像的坐标。此外，我们通过利用基于单个全息图的数值传播计算来估计这种线性关系，其中视场中一个物体的焦距是已知的。我们通过多个完全不同的实验案例证明了所提出的精确3D跟踪方法，即恢复海藻（tetraselmis）在水中的游泳路径以及在微振动刺激下卵巢癌细胞的快速重新聚焦。报告的实验结果验证了所提出的策略在动态测量和 3D 跟踪方面的有效性，而无需多次衍射计算和任何有关设置的精确知识。我们声称这是第一次将全息偏振复用设置本质上用于 3D 跟踪和/或快速准确的重新聚焦。 这意味着，根据我们的结果，除了样品的偏振分析之外，几乎任何偏振 DH 设置都可以保证沿光轴的精确聚焦，从而克服轴向分辨率差的限制。