MyoSight—semi-automated image analysis of skeletal muscle cross sections

Fig. 1. MyoSight identification of fiber borders, fiber-type, peri- and central nuclei. Representative images illustrating fiber border identification, fiber-type recognition, and peri- and central nuclei counting in the soleus from a WT mouse. a Representative image of original laminin stain in soleus of a WT mouse. b The FIJI “Find Edges” tool is used to enhance weak laminin staining. c Gaussian blurs are used to connect the breaks in the laminin staining separating adjacent fibers. d Fiber segmentation lines overlayed on original laminin stain. e Segmentation lines are colored to match laminin stain, and the image is flattened to enhance the laminin stain. f The flattened image is thresholded. g Individual regions of interest are created for each fiber. h All channels are combined for manual corrections of fiber borders and fiber-type. Representative images of fibers stained with antibodies to i MHC I, j MHC IIa, k MHC IIb, and l merged MHC immunofluorescent staining for all fiber-types in the soleus of a WT mouse with fiber borders overlaid. Representative MHC I/MHC IIa hybrid fiber defined by average pixel brightness for a fiber exceeds the threshold in two channels. m MHC I, n MHC…

Fig. 2. Manual corrections. a Representative image of incorrect analysis (Fiber 35) due to a discontinuation of laminin staining along the fiber border (arrow). b Manual correction of incorrectly defined fiber border in panel a (fiber 283). c Representative image of a muscle spindle (designated in this analysis as fiber 238) incorrectly identified as a myofiber. d Manual correction of incorrectly identified muscle spindle in panel c. e Representative image of interstitial region incorrectly identified as a myofiber (designated in this image as fiber 52). f Manual correction of incorrectly identified region in panel e. g Representative image of myofiber that was not identified. h Manual correction of unidentified myofiber in panel g (fiber 286). h Representative image of a single myofiber incorrectly identified as two fibers (designated 143 and 152 in this image). j Manual correction of incorrectly identified myofiber from panel (now fiber 242). Scale bars are 40 micron

Fig. 3. Inter-user reliability. Independent analyses of CSA as a function of fiber type by 4 users. Data from soleus of a WT mouse. a CSA of MHC I fibers in WT soleus, p > 0.05. b CSA of MHC IIa fibers from WT p > 0.05. c CSA of MHC IIx fibers from WT, p = 0.047. d Analyzed merged WT image from user 3. (Arrows indicate structures misidentified as fibers.) e Analyzed merged WT image from user 4. Scale bars are 50 microns

Fig. 4. CSA, fiber-type specific CSA, and fiber-type distribution. Comparisons of manual analysis to the analyses from MyoSight. a MHC I fibers from 3 WT and 3 mdx mice analyzed either manually (WT: n = 574 fibers, mdx: n = 572 fibers) or with MyoSight (WT: n = 664 fibers, mdx: n = 657 fibers). b MHC IIa fibers from 3 WT and 3 mdx mice analyzed either manually (WT: n = 1155 fibers, mdx: n = 1165 fibers) or with MyoSight (WT: n = 1039 fibers, mdx: n = 968 fibers). c MHC IIx fibers from 3 WT and 3 mdx mice analyzed either manually (WT: n = 193 fibers, mdx: n = 178 fibers) or with MyoSight (WT: n = 358 fibers, mdx: n = 358 fibers). d Correlation of CSA of MHC I fibers from WT and mdx mice from MyoSight with the CSAs of these fibers assessed manually. e Correlation of CSA of MHC IIa fibers from WT and mdx mice from MyoSight with the CSAs of these fibers assessed manually. f Correlation of CSA of MHC IIx fibers from WT and mdx mice from MyoSight with the CSAs of these fibers assessed manually. g Distribution of type I fibers analyzed manually as a function of binned CSA for WT and mdx fibers (n = 3, mean ± SEM). h Distribution of MHC IIa fibers analyzed manually as a function of binned CSA…

Fig. 5. Analyses of central and perinuclei in I, IIa, and IIx for WT and mdx mice. a The number of central nuclei in type I fibers from WT and mdx mice determined manually and with MyoSight. b The number of central nuclei in type IIa fibers from WT and mdx mice determined manually and with MyoSight. c The number of central nuclei in type IIx fibers from WT and mdx mice determined manually and with MyoSight. d The number of perinuclei in type I fibers from WT and mdx mice determined manually and with MyoSight. e The number of perinuclei in type IIa fibers from WT and mdx mice determined manually and with MyoSight. f The number of perinuclei in type IIx fibers from WT and mdx mice determined manually and with MyoSight. Differences between WT and mdx represented by * p ≤ 0.05,** p ≤ 0.01,*** p ≤ 0.001, and **** p ≤ 0.0001