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双螺旋点扩散函数(double-helixpointspreadfunction,DH-PSF)显微可实现纳米尺度的三维单颗粒示踪(three-dimensionalsingleparticletracking,3DSPT),被广泛应用于生命科学等领域。但是,DH-PSF显微的成像景深和定位精度有限,限制了其在活体厚样品中的应用,为了解决此问题,本文提出了一种基于轴向分光棱镜的多焦面DH-PSF显微(z-splitterprism-basedmultifocusDH-PSFmicroscopy,ZPMDM)方法和系统,通过将基于轴向分光棱镜的多焦面显微与DH-PSF相结合,在无需扫描的情况下提高DH-PSF显微的轴向定位范围和定位精度,解决完整活细胞内3DSPT的大景深探测难题。通过系统标定,ZPMDM中三个通道的平均三维定位精度分别为σ左(x,y,z)=(4.4nm,4.6nm,10.5nm),σ中(x,y,z)=(4.3nm,4.2nm,8.2nm),以及σ右(x,y,z)=(4.8nm,4.4nm,10.3nm),DH-PSF的有效景深扩展至6μm,实现了大景深范围内的甘油-水混合溶液中的荧光微球示踪,并初步研究了活巨噬细胞的吞噬现象,进一步验证了该方法的有效性,对于3DSPT的发展和应用具有重要意义。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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