Use of computer-assisted analysis for myofiber size measurements of rat soleus muscles from photographed images.

Since myofiber cross-sectional area measurements are important in describing myofiber adaptations to physiological and pathological changes, we developed a reproducible method for measuring myofiber size using fluorescent stains. Several 35-mm slides of dystrophin-, laminin-, and concanavalin A (ConA)-stained muscle sections were used to calculate myofiber cross-sectional areas and to compare different techniques and settings of an image capture system. Although variation in equipment settings did result in variation in myofiber area, the overall effect was of little practical significance (< 6%). Using midrange values for the settings of illumination, black level, and gain, reproducible quantitative data were collected and analyzed from 35-mm slides of FITC-labeled conA taken from atrophic, normal, and hypertrophic muscle samples. As expected, the atrophic muscle fibers were smaller. However, in hypertrophic muscle from compensatory overload, the fibers were composed of both large and small fibers. We found it important that the myofiber cross-sectional area measurements be expressed both in terms of average fiber areas and as frequency distribution histograms. In addition, detailed methodology of fiber area measurement must be provided.