Histological assessment of skeletal muscle tissue is commonly applied to many areas of skeletal muscle physiological research. Histological parameters including fiber distribution, fiber type, centrally nucleated fibers, and capillary density are all frequently quantified measures of skeletal muscle. These parameters reflect functional properties of muscle and undergo adaptation in many muscle diseases and injuries. While standard operating procedures have been developed to guide analysis of many of these parameters, the software to freely, efficiently, and consistently analyze them is not readily available. In order to provide this service to the muscle research community we developed an open source MATLAB script to analyze immunofluorescent muscle sections incorporating user controls for muscle histological analysis.

The software consists of multiple functions designed to provide tools for the analysis selected. Initial segmentation and fiber filter functions segment the image and remove non-fiber elements based on user-defined parameters to create a fiber mask. Establishing parameters set by the user, the software outputs data on fiber size and type, centrally nucleated fibers, and other structures. These functions were evaluated on stained soleus muscle sections from 1-year-old wild-type and mdx mice, a model of Duchenne muscular dystrophy. In accordance with previously published data, fiber size was not different between groups, but mdx muscles had much higher fiber size variability. The mdx muscle had a significantly greater proportion of type I fibers, but type I fibers did not change in size relative to type II fibers. Centrally nucleated fibers were highly prevalent in mdx muscle and were significantly larger than peripherally nucleated fibers.

The MATLAB code described and provided along with this manuscript is designed for image processing of skeletal muscle immunofluorescent histological sections. The program allows for semi-automated fiber detection along with user correction. The output of the code provides data in accordance with established standards of practice. The results of the program have been validated using a small set of wild-type and mdx muscle sections. This program is the first freely available and open source image processing program designed to automate analysis of skeletal muscle histological sections.