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Double-helix point spread function (DH-PSF) microscopy allows for nanoscale three-dimensional single particle tracking (3D SPT) and is commonly used in life sciences and other fields. However, its imaging depth-of-field (DOF) and localization accuracy are limited, which restricts its application in thick samples in vivo. To address this issue, this paper proposes a z-splitter prism-based multifocus DH-PSF microscopy (ZPMDM) method and system to improve the DOF and localization accuracy of DH-PSF microscopy without scanning. It solves the problem of large DOF detection of 3D SPT in whole living cells. By means of systematic calibration, the average 3D localization accuracies of the three channels of ZPMDM were determined to be σleft(x, y, z)=(4.4 nm, 4.6 nm, 10.5 nm), σmiddle(x, y, z)=(4.3 nm, 4.2 nm, 8.2 nm), and σright(x, y, z)=(4.8 nm, 4.4 nm, 10.3 nm). And the effective DOF of the system was extended to 6 μm. Furthermore, the ZPMDM system was used to track fluorescent microspheres in a glycerol-water mixture across a large depth-of-field range. The Brownian motion of the fluorescent microspheres in the mixture solution was also investigated. The experimental results demonstrate that the errors between the experimentally obtained diffusion coefficients and the theoretical diffusion coefficients are all within 10%. The reliability of the ZPMDM system in achieving single-particle 3D tracking imaging is verified by this study. The validity of the method was further verified by preliminarily investigating the phagocytosis phenomenon of live macrophages. This is significant for the development and application of nanoscale 3D SPT.
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Keywords:
- three-dimensional single particle tracking /
- double-helix point spread function /
- multifocus microscopy
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