Quantification of the 3D collagen network geometry in confocal reflection microscopy

The geometry of 3D collagen networks is a key factor that influences the behavior of live cells within extracellular matrices. This paper presents a hybrid, two-step method for fully automatic quantification of the 3D collagen network geometry at fiber resolution in confocal reflection microscopy images. First, a coarse binary mask of the entire network is obtained using steerable filtering and local Otsu thresholding. Second, individual collagen fibers are reconstructed by tracing maximum ridges in the Euclidean distance map of the binary mask. The proposed method, validated in a novel framework using 3D collagen gels with various collagen and Matrigel concentrations, reveal that Matrigel affects the collagen network geometry by decreasing the network pore size while preserving the fiber length and fiber persistence length.