Abstract
We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization. Using this approach, we analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells. Both applications indicate that the method can achieve 3D single-particle localization with a sub-pixel precision and sub-millisecond computation time. The precision is almost the same as that of the iterative nonlinear least-squares 3D Gaussian fitting method, but with two orders of magnitude higher computation speed. This approach can reduce considerably the time and costs for processing the large volume data of 3D images for 3D single-particle tracking, which is especially suited for 3D high-precision single-particle tracking, 3D single-molecule imaging and even new microscopy techniques.
Highlights
We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization
The intensity of the ‘‘wings’’ is dramatically reduced for confocal microscopy and the ‘‘body’’ is an approximate ellipsoid, which could be reliably characterized by the ratio of axial resolution (Rz) to lateral resolution (Rxy) (Rz=Rxy~3:28n=NA, where n is the refractive index of the objective medium and NA is the numerical aperture of the objective.)[34]
By scaling 3D images in axial direction according to the ratio, we can obtain the images with the intensity distribution of an approximate sphere
Summary
We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization Using this approach, we analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells. We analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells Both applications indicate that the method can achieve 3D single-particle localization with a sub-pixel precision and sub-millisecond computation time. These features make our algorithm to be a very competitive method for 3D SPT applications
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