Study on Measurement Method of Three-dimensional Position of Unlabeled Microspheres under Bright Background

Using computer vision technology to obtain the position and trajectory data of particle probe microspheres from microscope images has significance and value in the molecular field. However, most of the existing microsphere measurement methods are based on transmission, which can only be measured under transparent samples and substrates and are not suitable for the application scenario of living cell measurement. In this paper, a method based on reflectivity imaging is proposed to measure the three-dimensional position of the dark microspheres in the bright field. Based on the outermost ring radius method, the relationship between the inner ring radius of the microsphere spot and the out-of-focus distance was explored to measure the coordinates in the Z direction. Cardiomyocytes were combined with 10 um size silica microspheres. Experiments show that in a bright field with a high perturbation environment, it can achieve high precision measurement of dark microspheres and achieve three-dimensional position measurement with an accuracy of 50 nm in XY direction and 100 nm in Z direction.