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Supplementary materialsSegmentation correction frameworkThe correction was done by doing training on a manually segmented region (region A) and comparing this to the automatically segmented image on the same region. The corrected metrics (AVF and MVF) obtained were fitted and the correction factors were then applied to a second region on the automatic segmented image (region B). This segmentation was then validated with the ground truth of this region (region B). The segmentation correction framework is illustrated in Figure S1.Figure S1. Axon and myelin segmentation correction pipeline. Correction pipeline to compensate for the imperfect specificity and sensibility of AxonSeg, Top and bottom left: Two regions (region A and region B) were segmented both manually and automatically with AxonSeg. Example of myelin segmentation using AxonSeg and manual segmentation. Region A was used to train the corrections and Region B was used to validate the correction using a Bland-Altman analysis. Scale bar: 10 μm.2. Mean and std for the 6 metrics2.1 Axonal densityFGPSdSFCdCSTLSpRSTVFLatCC1251±66242±51204±51413±111220.9±81187±49201±53185±70C2240±60293±70180±53416±121259.9±85186±47211±57233±113C3291±84277±87189±60446±103281.2±96190±61210±64218±71C4235±60251±74168±44457±72263.0±60192±38204±59C5274±84244±80166±51445±121264.2±84226±68197±62C6229±78284±85160±58412±125232.9±80214±59199±65C7279±79258±97162±51482±130271.6±100252±97208±72C8258±107240±89158±54468±115239.3±111220±76211±70T1285±109297±111218±83501±127258.7±95230±78204±65T2240±92289±95241±87477±154275.8±78216±66205±59T3269±79316±91245±78554±115313.8±88220±52227±62T4286±93300±95237±76414±144275.2±81228±66198±62T5251±126282±107212±82481±161248.9±96234±65223±73T6241±107295±110235±82490±170282.9±110237±98208±86T7277±90300±89234±75514±130264.9±100226±94223±71T8257±103310±118241±79548±155310.0±119218±110226±105T9218±88268±106226±79413±191281.0±127245±111197±84T10224±111253±108227±74460±123289.4±108223±117198±68T11189±111221±123202±86451±131286.7±77228±78202±64T12222±66254±72223±57471±131277.5±90227±74202±56T13158±96196±75175±67355±122247.6±65234±55192±44L1171±76226±65184±60382±159277.1±80233±68223±60L2196±78240±68203±57394±154296.1±68271±62233±50L3162±49162±59156±54333±125249.9±61222±72194±53L4201±47194±44163±44397±108299.7±81306±88229±53L5187±61211±62182±55381±116275.6±79267±78229±66L6220±67310±101214±91453±202294.7±100303±88249±85S1189±53235±58173±53342±117264.2±64268±51196±49S2204±57258±65150±50283±97242.7±84271±86194±47S3161±49190±67152±49246±92298.1±85284±98231±57S4240±66316±91150±52213±46230.7±92246±77210±50Table S1. Axonal density (number of axons/50×50 μm?) values (mean ± std). FG= fasciculus gracilis, PSdS= postsynaptic dorsal column, FC= fasciculus cuneatus, dCST= dorsal corticospinal tract, LSp= lateral spinal nucleus, RST =rubrospinal tract, VF= ventral funiculus, and LatC= lateral cervical nucleus.2.2 Axon diameterFGPSdSFCdCSTLSpRSTVFLatCC11.44±0.151.39±0.181.28±0.201.17±0.081.17±0.171.33±0.161.32±0.151.24±0.21C21.43±0.181.38±0.121.38±0.251.18±0.121.15±0.171.37±0.261.32±0.171.16±0.26C31.46±0.191.42±0.191.42±0.231.13±0.091.10±0.141.27±0.221.29±0.211.16±0.17C41.36±0.181.37±0.131.41±0.151.16±0.091.09±0.091.30±0.101.29±0.15C51.39±0.121.38±0.131.40±0.141.12±0.111.09±0.151.22±0.111.29±0.16C61.37±0.321.38±0.241.47±0.421.20±0.191.11±0.261.21±0.161.32±0.26C71.40±0.211.37±0.191.44±0.351.07±0.101.08±0.231.18±0.221.26±0.23C81.36±0.301.40±0.311.44±0.341.15±0.151.07±0.261.27±0.221.28±0.21T11.41±0.231.36±0.201.43±0.291.09±0.131.18±0.231.27±0.111.32±0.19T21.37±0.201.36±0.111.34±0.131.08±0.111.18±0.111.30±0.121.31±0.18T31.38±0.231.37±0.181.32±0.151.09±0.141.12±0.131.29±0.091.23±0.15T41.47±0.211.38±0.161.39±0.191.13±0.121.20±0.151.31±0.141.36±0.23T51.45±0.271.34±0.211.33±0.231.08±0.141.10±0.221.26±0.161.25±0.22T61.50±0.381.40±0.261.36±0.241.09±0.161.14±0.201.21±0.271.34±0.34T71.33±0.231.24±0.181.23±0.171.04±0.121.08±0.191.13±0.241.21±0.21T81.35±0.241.27±0.141.27±0.201.03±0.091.09±0.191.15±0.311.19±0.27T91.44±0.311.40±0.171.34±0.221.17±0.161.06±0.251.14±0.311.24±0.33T101.44±0.341.33±0.231.30±0.21.09±0.131.08±0.191.06±0.251.20±0.21T111.37±0.301.35±0.211.28±0.181.09±0.111.10±0.131.15±0.201.22±0.24T121.48±0.311.38±0.231.30±0.211.08±0.141.07±0.151.17±0.171.22±0.21T131.32±0.501.37±0.281.36±0.221.11±0.111.09±0.121.17±0.121.30±0.12L11.35±0.421.35±0.301.32±0.291.08±0.101.02±0.131.11±0.191.21±0.20L21.24±0.161.22±0.151.21±0.110.97±0.121.00±0.091.06±0.131.19±0.10L31.42±0.241.43±0.231.36±0.221.13±0.111.09±0.111.14±0.111.33±0.17L41.29±0.101.29±0.081.27±0.141.04±0.101.00±0.141.10±0.141.24±0.12L51.34±0.221.37±0.221.33±0.221.08±0.151.03±0.151.11±0.131.27±0.19L61.26±0.151.21±0.161.22±0.180.96±0.141.00±0.161.06±0.151.15±0.17S11.34±0.161.32±0.161.21±0.201.04±0.101.02±0.111.11±0.111.26±0.14S21.37±0.141.26±0.081.33±0.201.14±0.151.03±0.171.11±0.131.31±0.17S31.19±0.221.20±0.181.30±0.291.08±0.170.99±0.201.07±0.161.24±0.15S41.24±0.241.12±0.071.27±0.281.21±0.121.06±0.161.14±0.161.25±0.15Table S2. Axon diameter (μm) values (mean ± std). FG= fasciculus gracilis, PSdS= postsynaptic dorsal column, FC= fasciculus cuneatus, dCST= dorsal corticospinal tract, LSp= lateral spinal nucleus, RST =rubrospinal tract, VF= ventral funiculus, and LatC= lateral cervical nucleus.2.3 G-ratioFGPSdSFCdCSTLSpRSTVFLatCC10.63±0.020.62±0.020.61±0.050.63±0.030.64±0.020.63±0.020.64±0.020.63±0.02C20.59±0.070.62±0.030.61±0.050.63±0.020.63±0.020.63±0.020.63±0.020.62±0.03C30.62±0.020.62±0.020.63±0.020.63±0.030.63±0.030.61±0.020.63±0.030.61±0.03C40.58±0.060.61±0.030.63±0.030.64±0.020.61±0.040.62±0.030.62±0.04C50.62±0.040.62±0.020.64±0.020.63±0.020.63±0.020.62±0.020.62±0.02C60.59±0.070.62±0.030.63±0.040.63±0.020.60±0.070.60±0.040.63±0.03C70.60±0.050.62±0.030.63±0.030.63±0.030.63±0.040.62±0.030.62±0.03C80.59±0.040.62±0.030.63±0.040.63±0.020.58±0.080.62±0.030.62±0.03T10.62±0.040.62±0.020.63±0.020.63±0.030.62±0.040.61±0.040.62±0.03T20.59±0.090.63±0.030.62±0.040.64±0.030.63±0.020.62±0.020.62±0.03T30.63±0.040.64±0.030.63±0.030.65±0.010.63±0.020.64±0.020.63±0.03T40.63±0.030.63±0.030.63±0.020.63±0.020.62±0.030.61±0.040.62±0.02T50.63±0.040.61±0.050.62±0.040.64±0.030.57±0.090.64±0.030.63±0.02T60.60±0.050.63±0.030.62±0.030.64±0.030.60±0.050.58±0.040.62±0.02T70.61±0.050.60±0.050.61±0.050.64±0.030.58±0.070.56±0.060.62±0.03T80.59±0.040.63±0.020.64±0.020.64±0.030.60±0.050.56±0.060.61±0.04T90.58±0.060.62±0.040.63±0.020.64±0.020.59±0.070.56±0.030.59±0.03T100.61±0.060.60±0.050.62±0.030.64±0.040.59±0.070.51±0.030.59±0.04T110.59±0.060.63±0.050.63±0.040.64±0.030.63±0.030.60±0.050.60±0.03T120.62±0.020.62±0.020.63±0.020.64±0.030.59±0.060.59±0.080.61±0.03T130.49±0.090.57±0.060.61±0.050.63±0.040.63±0.040.62±0.040.62±0.02L10.57±0.100.61±0.030.62±0.030.64±0.030.61±0.040.58±0.060.62±0.03L20.56±0.050.63±0.030.63±0.040.63±0.040.63±0.030.60±0.040.63±0.03L30.61±0.040.62±0.040.63±0.040.64±0.010.61±0.050.60±0.050.62±0.03L40.62±0.040.63±0.020.64±0.030.65±0.010.63±0.030.63±0.020.64±0.02L50.61±0.050.63±0.010.64±0.020.64±0.020.62±0.040.62±0.030.63±0.02L60.61±0.030.64±0.010.64±0.040.66±0.020.63±0.040.63±0.030.62±0.03S10.61±0.040.64±0.010.58±0.060.65±0.020.64±0.030.63±0.040.63±0.02S20.63±0.030.63±0.020.63±0.040.64±0.020.64±0.040.62±0.040.64±0.03S30.61±0.100.59±0.080.62±0.060.66±0.020.66±0.020.65±0.040.64±0.02S40.62±0.060.64±0.010.61±0.070.65±0.020.66±0.040.64±0.020.64±0.03 Table S3. G-ratio values (mean ± std). FG= fasciculus gracilis, PSdS= postsynaptic dorsal column, FC= fasciculus cuneatus, dCST= dorsal corticospinal tract, LSp= lateral spinal nucleus, RST =rubrospinal tract, VF= ventral funiculus, and LatC= lateral cervical nucleus.2.4 Myelin thicknessFGPSdSFCdCSTLSpRSTVFLatCC10.50±0.150.50±0.150.45±0.130.39±0.070.43±0.140.47±0.130.46±0.130.44±0.13C20.51±0.170.48±0.160.49±0.170.39±0.080.40±0.100.48±0.160.46±0.140.43±0.14C30.50±0.150.49±0.150.49±0.150.38±0.070.38±0.080.43±0.140.45±0.130.40±0.10C40.46±0.140.47±0.130.48±0.120.38±0.060.38±0.060.43±0.100.44±0.11 C50.48±0.140.47±0.120.48±0.110.37±0.060.38±0.070.41±0.090.44±0.11C60.47±0.170.48±0.170.50±0.200.41±0.120.40±0.130.41±0.120.47±0.17C70.50±0.180.49±0.180.50±0.180.37±0.070.39±0.110.42±0.120.44±0.15C80.45±0.130.47±0.130.48±0.140.37±0.050.36±0.080.42±0.090.43±0.09T10.48±0.170.47±0.160.49±0.160.38±0.080.41±0.110.42±0.100.46±0.13T20.47±0.140.46±0.110.45±0.110.36±0.060.40±0.080.43±0.100.45±0.13T30.44±0.100.43±0.090.41±0.070.34±0.030.36±0.030.39±0.040.39±0.05T40.51±0.190.48±0.150.48±0.160.38±0.070.42±0.120.45±0.140.47±0.16T50.51±0.170.46±0.140.46±0.140.36±0.070.38±0.120.42±0.130.43±0.13T60.52±0.210.48±0.180.48±0.170.36±0.080.40±0.120.42±0.130.47±0.19T70.44±0.150.42±0.120.41±0.110.35±0.060.37±0.080.38±0.100.41±0.13T80.44±0.130.41±0.100.42±0.120.34±0.070.38±0.100.39±0.140.41±0.14T90.51±0.220.48±0.180.46±0.180.41±0.150.38±0.140.40±0.170.44±0.21T100.50±0.190.46±0.170.45±0.150.36±0.070.37±0.100.35±0.110.41±0.13T110.48±0.160.47±0.140.44±0.130.37±0.080.38±0.090.39±0.110.42±0.15T120.50±0.150.47±0.150.45±0.150.36±0.080.37±0.090.40±0.110.42±0.14T130.47±0.210.49±0.160.49±0.140.38±0.070.38±0.070.40±0.070.44±0.10L10.46±0.160.45±0.140.44±0.160.36±0.070.37±0.090.38±0.110.42±0.14L20.40±0.070.39±0.060.39±0.030.32±0.030.33±0.020.34±0.030.37±0.03L30.49±0.150.48±0.130.46±0.120.38±0.080.38±0.070.40±0.080.45±0.13L40.41±0.050.40±0.030.40±0.050.34±0.030.34±0.030.35±0.040.39±0.05L50.46±0.140.47±0.150.46±0.140.38±0.100.37±0.090.38±0.090.43±0.13L60.39±0.070.38±0.070.40±0.080.32±0.050.34±0.040.35±0.040.37±0.07S10.46±0.110.45±0.120.41±0.120.36±0.070.37±0.070.39±0.070.43±0.10S20.49±0.140.43±0.080.47±0.130.40±0.090.37±0.080.39±0.070.45±0.12S30.40±0.100.39±0.080.43±0.110.36±0.050.35±0.060.37±0.080.40±0.07S40.420.36±0.030.45±0.090.40±0.050.37±0.060.38±0.060.41±0.07Table S4. Myelin thickness (μm) values (mean ± std). FG= fasciculus gracilis, PSdS= postsynaptic dorsal column, FC= fasciculus cuneatus, dCST= dorsal corticospinal tract, LSp= lateral spinal nucleus, RST =rubrospinal tract, VF= ventral funiculus, and LatC= lateral cervical nucleus.2.5 AVFFGPSdSFCdCSTLSpRSTVFLatCC10.22±0.030.22±0.040.19±0.050.22±0.040.17±0.030.21±0.030.21±0.030.16±0.05C20.21±0.040.23±0.020.20±0.040.23±0.040.17±0.030.20±0.040.21±0.030.15±0.04C30.23±0.020.23±0.030.21±0.040.23±0.040.16±0.020.18±0.050.19±0.040.15±0.03C40.19±0.040.22±0.040.21±0.040.25±0.020.15±0.030.17±0.040.19±0.05 C50.21±0.020.21±0.030.20±0.030.23±0.030.15±0.030.17±0.030.18±0.04C60.19±0.040.22±0.040.21±0.050.24±0.030.13±0.030.16±0.040.19±0.05C70.22±0.030.22±0.040.21±0.050.22±0.030.15±0.030.17±0.030.18±0.05C80.21±0.070.23±0.060.21±0.060.25±0.030.14±0.040.17±0.040.19±0.04T10.22±0.040.23±0.040.22±0.050.23±0.020.15±0.030.18±0.040.19±0.04T20.19±0.060.22±0.040.22±0.050.22±0.040.17±0.020.19±0.030.19±0.03T30.21±0.050.23±0.050.22±0.040.26±0.030.18±0.030.19±0.030.19±0.04T40.23±0.050.22±0.030.23±0.030.21±0.040.17±0.020.19±0.030.20±0.04T50.20±0.070.21±0.060.20±0.060.22±0.050.14±0.040.19±0.040.19±0.04T60.19±0.030.22±0.030.21±0.040.22±0.040.16±0.030.18±0.050.20±0.05T70.19±0.030.19±0.030.19±0.040.22±0.040.15±0.040.16±0.050.19±0.05T80.19±0.040.21±0.040.21±0.040.22±0.040.17±0.040.18±0.080.18±0.06T90.19±0.050.21±0.050.21±0.040.21±0.070.15±0.040.18±0.050.19±0.06T100.19±0.070.19±0.060.20±0.060.21±0.040.16±0.040.15±0.060.18±0.05T110.15±0.070.17±0.070.18±0.060.21±0.030.15±0.020.16±0.040.18±0.05T120.20±0.040.21±0.030.20±0.040.22±0.040.14±0.030.16±0.040.18±0.05T130.16±0.090.19±0.060.18±0.060.19±0.040.14±0.030.16±0.030.19±0.04L10.17±0.060.19±0.040.18±0.040.19±0.060.14±0.020.15±0.030.18±0.04L20.16±0.050.17±0.040.17±0.040.17±0.070.14±0.030.16±0.030.18±0.03L30.19±0.040.18±0.040.17±0.050.19±0.040.14±0.020.14±0.030.19±0.04L40.18±0.030.17±0.020.16±0.030.20±0.030.14±0.020.18±0.030.19±0.03L50.18±0.040.20±0.030.18±0.040.20±0.020.14±0.020.16±0.020.20±0.03L60.19±0.030.20±0.030.16±0.040.17±0.030.14±0.020.16±0.020.18±0.03S10.19±0.040.21±0.040.16±0.040.17±0.030.13±0.020.15±0.020.18±0.03S20.20±0.030.20±0.030.17±0.040.18±0.020.12±0.020.15±0.030.19±0.03S30.15±0.060.16±0.060.17±0.070.15±0.030.14±0.020.15±0.030.19±0.03S40.19±0.070.17±0.030.17±0.060.17±0.050.12±0.040.14±0.040.18±0.03Table S5. AVF values (mean ± std). FG= fasciculus gracilis, PSdS= postsynaptic dorsal column, FC= fasciculus cuneatus, dCST= dorsal corticospinal tract, LSp= lateral spinal nucleus, RST =rubrospinal tract, VF= ventral funiculus, and LatC= lateral cervical nucleus.2.6 MVFFGPSdSFCdCSTLSpRSTVFLatCC10.34±0.050.36±0.070.28±0.070.33±0.050.25±0.040.32±0.050.31±0.050.25±0.07C20.33±0.090.37±0.070.30±0.070.34±0.040.27±0.040.31±0.070.32±0.050.24±0.07C30.37±0.060.36±0.060.31±0.070.34±0.050.24±0.050.28±0.090.29±0.070.23±0.07C40.29±0.090.33±0.070.30±0.070.36±0.050.23±0.060.27±0.080.28±0.08 C50.33±0.050.33±0.040.30±0.050.34±0.030.23±0.050.28±0.050.28±0.07C60.28±0.100.35±0.070.29±0.070.36±0.060.20±0.060.25±0.070.29±0.08C70.35±0.090.36±0.080.31±0.070.34±0.070.22±0.060.26±0.060.27±0.08C80.31±0.120.34±0.080.30±0.070.37±0.030.20±0.070.27±0.060.29±0.06T10.33±0.080.37±0.080.32±0.080.35±0.060.23±0.060.28±0.070.30±0.07T20.29±0.120.34±0.070.32±0.090.32±0.050.27±0.040.29±0.050.30±0.06T30.31±0.090.33±0.080.32±0.050.35±0.040.26±0.050.28±0.050.28±0.05T40.36±0.060.34±0.060.35±0.060.31±0.050.27±0.060.30±0.060.30±0.06T50.31±0.120.31±0.110.30±0.100.32±0.060.21±0.100.28±0.080.29±0.06T60.30±0.070.34±0.070.32±0.070.33±0.050.25±0.060.28±0.090.30±0.07T70.29±0.070.29±0.070.28±0.080.32±0.060.22±0.090.23±0.090.28±0.07T80.27±0.060.30±0.050.29±0.050.31±0.040.25±0.080.26±0.140.26±0.09T90.30±0.100.33±0.090.31±0.070.30±0.080.23±0.080.28±0.110.28±0.11T100.29±0.130.30±0.130.29±0.100.31±0.070.24±0.080.23±0.120.26±0.08T110.24±0.120.26±0.120.26±0.100.31±0.060.24±0.060.24±0.100.27±0.09T120.32±0.060.33±0.060.31±0.070.32±0.050.23±0.080.26±0.100.28±0.08T130.25±0.170.29±0.110.27±0.110.28±0.070.22±0.070.26±0.080.29±0.06L10.27±0.100.30±0.060.27±0.060.27±0.070.22±0.060.24±0.080.28±0.06L20.24±0.090.26±0.080.24±0.070.23±0.110.19±0.050.23±0.060.26±0.04L30.29±0.070.26±0.070.25±0.080.28±0.060.21±0.050.22±0.070.29±0.06L40.27±0.040.26±0.040.23±0.050.28±0.040.20±0.040.26±0.050.28±0.04L50.27±0.070.31±0.050.26±0.060.29±0.050.21±0.050.24±0.060.30±0.05L60.27±0.050.29±0.040.22±0.050.24±0.040.19±0.040.23±0.040.25±0.05S10.28±0.060.31±0.070.22±0.080.24±0.050.20±0.050.24±0.050.26±0.05S20.30±0.050.30±0.040.23±0.070.26±0.030.17±0.030.22±0.030.27±0.05S30.19±0.090.22±0.090.22±0.100.19±0.040.18±0.030.21±0.040.26±0.04S40.27±0.100.26±0.020.22±0.090.24±0.060.16±0.060.20±0.060.25±0.05Table S6. MVF values (mean ± std). FG= fasciculus gracilis, PSdS= postsynaptic dorsal column, FC= fasciculus cuneatus, dCST= dorsal corticospinal tract, LSp= lateral spinal nucleus, RST =rubrospinal tract, VF= ventral funiculus, and LatC= lateral cervical nucleus.3. Effect of fixation protocolWe varied slightly the concentration of the fixatives and the type of fixation (immersion and perfusion) (see Table S7). Measures of circularity (roundness of the axon), ellipticity (flattening of the axon), solidity (compactness of the axon) and g-ratio were reported for two different ROI (FG and VST tracts) to quantify the effect of fixation protocols on the WM microstructure integrity (see Figure S2). No significant differences were found between protocols on the microstructure integrity. Type of fixationPFA (%)Ga (%)Osmification (2%) duration (h)Acetone baths (%)Protocol #1perfusion33510, 35, 50, 70, 75, 85, 90, 100Protocol #2perfusion22.5510, 25, 35, 50, 75, 85, 90, 100Protocol #3immersion4- 510, 25, 35, 50, 75, 85, 90, 100Protocol #4immersion42610, 25, 35, 50, 75, 85, 90, 100Protocol #5immersion42.5610, 25, 35, 50, 75, 85, 90, 100Protocol #6immersion42410, 25, 35, 50, 75, 100Protocol #7immersion4 -250, 75, 100Table S7. Protocols of tissue fixation used to assess the effect of the different parameters on the tissue integrity. Seven protocols with different parameters were explored: type of fixation (immersion or perfusion), concentration of fixatives (PFA= paraformaldehyde, Ga= glutaraldehyde), osmification duration and number of acetone graded baths.Figure S2. Effect of different fixation protocols on the myelin integrity. a: SEM images of two different regions for each protocol, b: full slice SEM image identifying FG and VST regions, c: schematic illustration of axon shape factors (circularity, solidity and ellipticity), d: Mean and std values of axon shape factors and g-ratio in FG tract (left graph) and in VST tract (right graph). Scale bar: 2 μm. ................
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