Measurement of Orientation and Distribution of Cellular Alignment and Cytoskeletal Organization WILLIAM J. KARLON, 1 PIN-PIN HSU, 1 SONG LI, 1 SHU CHIEN, 1 ANDREW D. MCCULLOCH, 1 and JEFFREY H. OMENS 2 1 Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, Mail Code 0412, La Jolla, CA and 2 Department of Medicine, University of California, San Diego, 9500 Gilman Drive, Mail Code 0613-J, La Jolla, CA (Received 22 December 1998; accepted 23 August 1999) Abstract—Endothelial cells elongate and align with the direc- tion of applied fluid shear stress. Previously, automated meth- ods for analysis of cell orientation distribution have used Fourier- or fractal-based methods. We used intensity gradients in images of control and sheared endothelial cells to measure orientation distributions. Automated measurements of mean ori- entation and angular deviation compared favorably with manual measurements. There was a significantly greater angular devia- tion in images of control cells compared with sheared cells. Automated methods were also used to quantify organization of cytoskeletal fibers using the local angular deviation and a mea- sure of the local coalignment of fibers called the coalignment ratio. The local angular deviation of microtubules and mi- crofilaments was significantly smaller in sheared cells com- pared with control. The coalignment of cytoskeletal fibers was significantly greater in sheared cells. We conclude that image intensity gradients can be used rapidly, accurately, and objec- tively to measure cell orientation distributions and cytoskeletal filament organization. © 1999 Biomedical Engineering Soci- ety. S0090-69649900506-8 Keywords—Endothelial cells, Shear stress, Intensity gradients, Actin filaments, Microtubules. INTRODUCTION The orientation response of cells to mechanical stimuli such as stretch and shear stress has been de- scribed in several cell types. 2,3,17 In cell culture models, endothelial cells respond by elongation and alignment in the direction of applied steady flow. 3,4,11 Early reports on this orientation response were mostly qualitative. Later, manual measurements and automated computer methods were used to quantify the response of individual cells. 15 Cytoskeletal remodeling in response to fluid shear has been described qualitatively, 12,16,19 but presently, few quantitative measurements of cytoskeletal organization have been performed. Previous investigations have used Fourier methods 9,14 and fractals 18,20 to analyze cytoskel- etal structure, but the accuracy of these methods is dif- ficult to test. Cytoskeletal elements such as actin mi- crofilaments have been analyzed in a wound healing study using classification into categories such as parallel arrangement or lack of central filaments. 10 This type of semiquantitative method provides descriptive information about organization, but can be difficult to interpret and analyze statistically. A recent quantitative study of en- dothelial cell cytoskeleton reorganization by Galbraith et al. used manual measurement techniques and indicated that remodeling occurs in a time-dependent manner. 7 Au- tomated techniques could be used to quantify time- dependent cell alignment in response to shear stress or cytoskeletal remodeling rapidly and objectively. A quan- titative study of actin stress fiber orientation was per- formed by Petroll et al. using Fourier methods. 14 They describe an orientation index based on the power spec- trum of the two-dimensional Fourier transform. A similar approach was used by Palmer and Bizios for quantifying endothelial cell alignment, but they concluded that Fou- rier methods were unable to predict angular deviation accurately. 13 Image analysis techniques developed for assessing oriented texture patterns 1 were shown recently to be use- ful for measuring myofiber disarray associated with fa- milial hypertrophic cardiomyopathy. 8 We modified these techniques to estimate rapidly, accurately, and objec- tively the mean, angular deviation and distribution of orientation of endothelial cells which became aligned in response to shear stress. We also showed that intensity gradients can be used to quantify the organization of cytoskeletal filaments. For this study, cytoskeletal orga- nization is defined to be the degree to which cytoskeletal fibers in a local region have the same orientation. Local is defined by a length scale that is small with respect to the maximum dimension of the cell, so as to describe the behavior of individual fibers rather than the cell as a whole. We hypothesized that measurement of local con- Address correspondence to Jeffrey H. Omens, Department of Medi- cine, University of California, San Diego, 9500 Gilman Drive, Mail Code 0613-J, La Jolla, CA 92093. Electronic mail: omens@be- research.ucsd.edu Annals of Biomedical Engineering, Vol. 27, pp. 712–720, 1999 0090-6964/99/276/712/9/$15.00 Printed in the USA. All rights reserved. Copyright © 1999 Biomedical Engineering Society 712