Zheng Juanjuan, Gao Peng, Shao Xiaopeng, Nienhaus G Ulrich
Appl Opt. 2017 Nov 10;56(32):9000-9005. doi: 10.1364/AO.56.009000.
Opposed-view digital holographic microscopy (OV-DHM) with autofocusing and out-of-focus background suppression was demonstrated and applied to measure the refractive index (RI) of suspended HeLa cells. In OV-DHM, a specimen is illuminated from two sides in a 4π-like configuration. The generated two opposite-view object waves, which have orthogonal polarization orientations, interfere with a common reference wave, and the generated holograms are recorded by a CMOS camera. The image plane of the sample was determined by finding the minimal variation between the two object waves. The out-of-focus background was suppressed by averaging the two object waves. Simultaneous determination of both the cell thickness and the phase retardation was avoided by using a spheroidal model for the detached cell obtained from confocal microscopy. Thus, the RI of suspended HeLa cells was measured from phase images of OV-DHM, with the thickness of the cells estimated by using a constant axial-to-lateral ratio. This measurement strategy reveals the RI with an accuracy of ∼10% of the RI difference between cells and surrounding medium.
展示了具有自动聚焦和离焦背景抑制功能的对视图数字全息显微镜（OV-DHM），并将其应用于测量悬浮的HeLa细胞的折射率（RI）。在OV-DHM中，样本以类似4π的配置从两侧进行照明。产生的具有正交偏振方向的两个对视图物体波与一个公共参考波干涉，产生的全息图由CMOS相机记录。通过找到两个物体波之间的最小变化来确定样品的像平面。通过对两个物体波进行平均来抑制离焦背景。通过使用从共聚焦显微镜获得的分离细胞的球体模型，避免了同时确定细胞厚度和相位延迟。因此，从OV-DHM的相位图像测量悬浮HeLa细胞的RI，并使用恒定的轴向与横向比率估计细胞厚度。这种测量策略揭示的RI的精度约为细胞与周围介质之间RI差异的10%。
