Analyses on cytoskeleton structure basing on confocalmicroscope

Actin microfilaments are the main components of cytoskeleton. Some previous studies have shown that the state of cell adhesion regulates the biological behavior of stem cells such as differentiation and apoptosis by affecting the structure of microfilaments. The primary method of observing the structure of microfilaments is using laser confocal microscopy to obtain fluorescence images of microfilament, but usually only the two-dimensional structure of microfilaments was analyzed, and few studies have focused on the three-dimensional structure of microfilaments. In this paper, we proposed a three-dimensional reconstruction method of microfilaments based on laser confocal microscopy. The point spread function of a confocal microscope was fitted by three-dimensional Gaussian function, and then we got the Gaussian distribution of the z -axis light intensity by decoupling the x - y direction with the z direction of the function. Subsequently, the geometric parameters of the major fibers in a cell were obtained through image processing methods, and then the three-dimensional geometric information of the microfilament was extracted by using fitting calculation, density clustering, ellipsoid fitting and other operations to deal with filament images. In addition, we constructed circular and branch-shaped micropatterned substrates with an area of 1256 μm2, and we adopted the three-dimensional reconstruction method established in this study to reconstruct and analyze the microfilaments of bone marrow mesenchymal stem cells, which were cultured on the micropatterned substrates. The results showed that the spatial distribution of geometric parameters such as the orientation of the microfilaments, the number of intersection, the volume, and the aspect ratio for the cells with two shapes were significantly different, indicating that the mechanical state of the cells was influenced by the spreading shape. The purpose of this study is to establish a method for extracting the three-dimensional structural parameters of microfilaments, which can provide a basis for revealing the mechanism of mechanotransduction inside cells.