Measuring the regulation of keratin filament network dynamics.

The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

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.