MyelTracer: A Semi-Automated Software for Myelin g-Ratio Quantification

MyelTracer semi-automatically traces axons and myelin and returns results consistent with manual tracing. A–D, Electron micrograph of dorsal corpus callosum, software-generated threshold overlays, and postselection overlay (blue) show different steps of the image analysis. * periaxonal space. E, Electron micrograph with MyelTracer overlay and outlines for features including axon diameter (a), inner myelin diameter (d), and outer myelin diameter (D), each computed from the respective areas. F, Schematic for g-ratio scatterplot illustrating how MyelTracer calculates the g-ratio. G, Image of the main user interface of MyelTracer. H, Schematic showing an example workflow using MyelTracer. I, Representative electron micrograph of corpus callosum from one-month-old wild-type mice. J, G-ratio scatter plot from manual and MyelTracer quantifications for the same axons. ANCOVA test p = 0.8996 (slopes) and p = 0.7044 (intercepts), n = 174 axons. K, G-ratio from manual and MyelTracer quantification paired for the same axons. Student’s t test p = 0.9045, n = 174 axons. L, Comparison of time consumption between manual quantification and quantification using MyelTracer. Individual data points represent the average time spent per axon per image; p = 0.0069; Student’s t test. Scale bar: 1 μm. M–P, Illustration of MyelTracer’s ability to quantify myelin sheets with folds (arrowheads). Folds require the user to draw a white line (arrows) with the cut tool to connect the area inside the fold with the outside area for semi-automated detection. Scale bar: 1 μm. ns, no significance. **p < 0.01.

MyelTracer accurately returns g-ratios for myelinated tissues of varying axonal density. A, Representative electron micrograph of the optic nerve at two months of age (2 M) with MyelTracer-generated overlays (blue). Scale bar: 1 μm. B, g-Ratio scatter plot from manual and MyelTracer quantifications for the same axons. ANCOVA test p = 0.8311 (slopes) and p = 0.4695 (intercepts), n = 193 axons. C, g-Ratios from manual and MyelTracer quantifications paired for the same axons. p = 0.3406, n = 193 axons. D, Comparison of time consumption between manual quantification and quantification using MyelTracer. Individual data points represent the average time spent per axon per image; p = 0.0046. E, Representative electron micrograph of the sciatic nerve at one month of age with MyelTracer-generated overlays (blue). Scale bar: 2 μm. F, g-Ratio scatter plot from manual and MyelTracer quantifications for the same axons. ANCOVA test p = 0.7437 (slopes) and p = 0.8997 (intercepts), n = 104 axons. G, g-Ratios from manual and MyelTracer quantifications paired for the same axons. p = 0.9146, n = 104 axons. H, Comparison of time consumption between manual quantification and quantification using MyelTracer. Individual data points represent the average time spent per axon per image; p < 0.0001; Student’s t test. ns, no significance. **p < 0.01, ***p < 0.001.