Abstract
BackgroundManual analysis of cross-sectional area, fiber-type distribution, and total and centralized nuclei in skeletal muscle cross sections is tedious and time consuming, necessitating an accurate, automated method of analysis. While several excellent programs are available, our analyses of skeletal muscle disease models suggest the need for additional features and flexibility to adequately describe disease pathology. We introduce a new semi-automated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses.ResultsWe describe staining and imaging methods that generate high-quality images of immunofluorescent-labelled cross sections from mouse skeletal muscle. Using these methods, we can analyze up to 5 different fluorophores in a single image, allowing simultaneous analyses of perinuclei, central nuclei, fiber size, and fiber-type distribution. MyoSight displays high reproducibility among users, and the data generated are in close agreement with data obtained from manual analyses of cross-sectional area (CSA), fiber number, fiber-type distribution, and number and localization of myonuclei. Furthermore, MyoSight clearly delineates changes in these parameters in muscle sections from a mouse model of Duchenne muscular dystrophy (mdx).ConclusionsMyoSight is a new program based on an algorithm that can be optimized by the user to obtain highly accurate fiber size, fiber-type identification, and perinuclei and central nuclei per fiber measurements. MyoSight combines features available separately in other programs, is user friendly, and provides visual outputs that allow the user to confirm the accuracy of the analyses and correct any inaccuracies. We present MyoSight as a new program to facilitate the analyses of fiber type and CSA changes arising from injury, disease, exercise, and therapeutic interventions.
Highlights
Accurate measurements of cross-sectional area (CSA), fiber-type distribution, and myonuclei number and location provide critical information needed to evaluate the consequences of disease and injury and to evaluate the efficacy of therapeutic interventions and/or exercise in improving skeletal muscle function [1]
We describe a new semi-automated image analysis tool called MyoSight, which operates as a FIJI plugin
Overview and use of MyoSight The MyoSight program functions as a plugin for FIJI, a freely available image analysis platform produced by the National Institutes of Health
Summary
Accurate measurements of cross-sectional area (CSA), fiber-type distribution, and myonuclei number and location provide critical information needed to evaluate the consequences of disease and injury and to evaluate the efficacy of therapeutic interventions and/or exercise in improving skeletal muscle function [1]. These measurements, when completed manually, are time consuming and prone to user error and bias. There are several programs currently available for histological segmentation of muscle fibers to quantify CSA, fiber-type distribution, perinuclei (nuclei along the perimeter of fiber), and central nuclei [2,3,4,5,6,7,8,9,10,11]. We introduce a new semiautomated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses
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