3D segmentation of keratin intermediate filaments in confocal laser scanning microscopy

In this paper, we propose and compare different methods for the 3D segmentation of keratin intermediate filaments (KFs) in images acquired using confocal laser scanning microscopy (CLSM). KFs are elastic cables forming a complex scaffolding within epithelial cells. They are involved in many basic cell functions. To understand the mechanisms of filament formation and network organisation under physiological and pathological conditions, quantitative measurements of dynamic network alterations are essential. Segmenting KFs is a key component for analyzing their dynamic and biomechanical properties. KFs were labeled with fluorescent keratins to allow high resolution imaging of network dynamics in native cells. Our segmentation methods follow the principle of ridge enhancement filtering and subsequent centerline extraction. The evaluation of the methods is two-fold: (i) We develop synthetic data that exhibit the characteristics of real CLSM data to evaluate the precision of the different methods in terms of centerline localisation and (ii) we perform a connected component analysis on the segmentation results of real KF data to assess whether the connectivity of highly complex networks is being preserved by the segmentation. Our evaluation shows that in the presence of strong noise and despite the highly anisotropic spatial resolution of CLSM images the proposed method is able to accurately localize the centerlines of the KFs and to preserve the KF networks´ connectivity. Taken together this is a strong indicator that also the network topology is being preserved.