Sperm Flagellum Center-Line Tracing in Fluorescence 3D+t Low SNR Stacks Using an Iterative Minimal Path Method

Abstract

Intracellular calcium ([Ca\(^{2+}\)]i) regulates sperm motility. Visualizing [Ca\(^{2+}\)]i in 3D is not a simple matter since it requires complex fluorescence microscopy techniques where the resulting images have very low intensity and consequently low SNR (Signal to Noise Ratio). In 3D+t sequences, this problem is magnified since the flagellum beats (for human sperm) at an average frequency of 15 Hz, making it harder to obtain the three dimensional information. Moreover, 3D holographic techniques do not work for these fluorescence based images. In this paper, an algorithm to extract the flagellum’s center-line in 3D+t stacks is presented. For this purpose, an iterative algorithm based on the fast-marching method is proposed to extract the flagellum’s center-line. Quantitative and qualitative results are presented in a 3D+t stack to demonstrate the ability of the proposed algorithm to trace the flagellum’s center-line. Our method was qualitative and quantitatively compared against state-of-the-art tubular structure center-line extraction algorithms outperforming them and reaching a Precision and Recall of 0.96 as compared with a semi-manual method used as reference. The proposed methodology has proven to solve a major problem related with the analysis of the 3D motility of sperm cells in images with very low intensity.