Combined with SHG imaging, the GLCM approach has been used to quantitatively analyze the collagen fibers.4,35 But it simply classifies different biological tissues by the feature values without providing any information directly associated with the geometrical arrangement of collagen fibrillar bundles. Generally, GLCM features along one or two of the four specific directions of 0 deg, 45 deg, 90 deg, and 135 deg or average feature values along these four directions are extracted to quantitatively analyze the SHG images.4,29,31,32,36 However, the dominant orientation of the collagen fibers is usually ignored in the conventional GLCM analysis. Since the orientation is an important characteristic of collagen fibers with a filamentous structure, the GLCM features calculated along the dominant orientation of collagen fibers are different from those calculated along the other directions. As a result, the texture information obtained from the feature curves is dependent on the direction selected in GLCM analysis. By combining the dominant orientation of collagen fibers into the GLCM analysis, the GLCM feature curves calculated may provide more information for detailed morphological characterization of the collagen fibers, thus leading to further sights into various physiological and pathological processes, such as the structural modification of the extracellular matrix during the migration and invasion of tumor cells.