Figure 2. Method for morphometric analysis of myofibers.

(A) Hematoxylin and eosin stained intercostal muscle cross sections were imaged with light microcopy at a 200x magnification. (B) Fiji^TM or Photoshop^TM imaging software were used to generate a black and white segmented mask outlining the boundaries of each myofiber. (C) In Photoshop^TM, the mask was overlaid on the gray-scaled original image, transparency increased, and manual corrections were made to define the boundaries of each fiber. Once boundaries are defined, the corrected mask is thresholded in Fiji^TM, and a particle analysis tool was used to obtain measurements. Measurements include minor axis length, fiber cross sectional area, and roundness. (D) The minor axis is identified as the shortest axis of a best fit ellipse (Arrow). Pixels of this axis and of the cross sectional areas were converted to μm and μm² respectively for each myofiber. The percent of angulated fibers (example labeled with “a” in insert of panel C) were determined manually by a masked evaluator.