Abstract
Double-helix point spread function (DH-PSF) microscopy can realize three-dimensional single particle tracking (3D SPT) on a nanoscale, and is widely used in life sciences and other fields. However, its imaging depth-of-field (DOF) and localization accuracy are limited, which hinders 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 three channels of ZPMDM are determined to be <i>σ</i><sub>L(<i>x, y, z</i>)</sub> = (4.4 nm, 4.6 nm, 10.5 nm), <i>σ</i><sub>M(<i>x, y, z</i>)</sub> = (4.3 nm, 4.2 nm, 8.2 nm), and <i>σ</i><sub>R(<i>x, y, z</i>)</sub> = (4.8 nm, 4.4 nm, 10.3 nm). And the effective DOF of the system is extended to 6 μm. Furthermore, the ZPMDM system is 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 is also investigated. The experimental results demonstrate that the errors between the experimentally obtained diffusion coefficients and the theoretically calculated diffusion coefficients are all within 10%. The reliability of the ZPMDM system in achieving single-particle 3D tracking imaging is verified in this study. The validity of the method is further verified by preliminarily investigating the phagocytosis phenomenon of live macrophages. It is of significance for the development and application of nanoscale 3D SPT. The ZPMDM system is shown in the attached figure.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.