‘NMJ-morph’ User Guide This user guide comprises a workflow that has been designed for use in conjunction with Figure 2 (and Supplementary Figures 1 and 2) of the manuscript and the downloadable spreadsheets and image bank. The following descriptions guide the user through the stages of analysis for each NMJ using ImageJ (or Fiji), and correspond in order to the workflow depicted in Figure 2 of the manuscript. Instructions in italics refer to the selectable functions in the ImageJ menus. Variables listed in red typeface are defined as ‘core variables’, those in blue as ‘derived variables’ and those in green as ‘associated nerve and muscle variables’. See manuscript for further explanation and definition of individual terms. Basic Image Details Before entering numerical values into the spreadsheet for any of the NMJ variables, the basic properties of the image must be noted first. This is the frame size of the image, which must be recorded as both length in pixels and length in metric units. For the sample NMJs provided in the image bank, the frame size is 512×512 pixels and 67.48×67.48 μm (entered into the relevant cells as ‘512’ and ‘67.48’). Initial Image Processing ImageJ Operations Open maximum intensity projection in ImageJ Image Colour Split channels. Then, for each channel separately Image Adjust Threshold (select ‘Huang’, ‘Yen’, or adjust threshold manually with top slider. To allow accurate threshold selection, a second maximum intensity projection should be opened for comparison. Once the threshold has been selected, close the window at the top left – don’t select ‘Apply’) Process Noise Despeckle Select background colour using colour picker tool, then manually erase extraneous background detail with paintbrush tool (to isolate the relevant NMJ on a white background) Process Binary Make binary (Keep all options selected – the resulting binary image will have either a red or green background. Selecting Make binary for a second time at this stage creates a white background; the coloured backgrounds are required however for ‘overlap’ to be calculated – see later) Save the ‘cleaned’ pre- and post-synaptic images separately (as tiff files). All subsequent analysis is performed on these images (with the exception of the ‘segmented particles’ function – see number of AChR clusters, average area of AChR clusters, ‘fragmentation’). Presynaptic Measurements Number of Axonal Inputs ImageJ Operations The original maximum intensity projection should be inspected for any evidence of polyneuronal innervation and the number of individual axonal inputs per NMJ noted in the spreadsheet. Axon Diameter ImageJ Operations Open saved ‘clean’ image Measure axon diameter with line tool at points of maximum and minimum diameter, and at edge of endplate (this latter measurement should be either the point at which the axon crosses the edge of the endplate, or the point at which the axon ramifies, if this lies outside the area of the endplate) Results Summarize Record mean diameter in spreadsheet. Nerve Terminal Perimeter Nerve Terminal Area ImageJ Operations Open saved ‘clean’ image Select background colour with colour picker tool, then manually erase axon with paintbrush tool Edit Selection Create selection (this will automatically outline the remaining nerve terminals) Analyze Measure Record area & perimeter in spreadsheet. Number of Terminal Branches (n) Number of Branch Points Total Length of Branches (l) Average Length of Branches (l/n) ‘Complexity’ ImageJ Operations Open saved ‘clean’ image Process Binary Make binary (background becomes white) Process Binary Skeletonize Select background colour with colour picker tool, then manually erase axon with paintbrush tool Plugins BinaryConnectivity (keep ‘white particles on black background’ selected – the binary image then ‘disappears’ – this is correct) Analyze Histogram List Enter the count for values 0, 2, 4 & 5 into the spreadsheet Branch analysis will be performed automatically for the above 5 variables. Postsynaptic Measurements AChR Perimeter AChR Area ImageJ Operations Open saved ‘clean’ image Edit Selection Create selection (this will automatically outline the AChR clusters) Analyze Measure Record area & perimeter in spreadsheet Continue analysis below. Endplate Diameter ImageJ Operations (Continued from above) Process Subtract background Select ‘Create background’ (keep ‘rolling ball radius’ at 50.0 pixels) Process Binary Make binary (background becomes white and edge of endplate becomes sharper) Measure maximum endplate diameter with line tool Record maximum diameter in spreadsheet Continue analysis below. Endplate Perimeter Endplate Area ImageJ Operations (Continued from above) Edit Selection Create selection (this will automatically outline the endplate) Analyze Measure Record area & perimeter in spreadsheet. ‘Density’ ImageJ Operations ‘Density’ is calculated automatically once the values for AChR cluster area and endplate area have been entered into the spreadsheet. ‘Area of Synaptic Contact’ ‘Overlap’ ImageJ Operations Open saved ‘clean’ images of both nerve terminals and endplate. For endplate Edit Invert. With both images now open Image Stacks Tools Concatenate (select ‘OK’ – images will be merged into one stack) Image Stacks Z project (keep ‘average intensity’ selected – a 3 colour image will be produced in black and 2 tones of either red or green, as in Figure 2) Process Binary Make binary (this will leave just the unoccupied AChRs) Edit Selection Create selection (this will automatically outline the area of unoccupied AChRs) Analyze Measure Enter this value into the spreadsheet and the ‘area of synaptic contact’ and ‘overlap’ and will be calculated automatically. Number of AChR Clusters Average Area of AChR Clusters ‘Fragmentation’ ImageJ Operations Open original maximum intensity projection (not the ‘clean’ image) Image Colour Split channels and select endplate channel Process Binary Make binary Process Find maxima Select ‘Segmented particles’ (keep ‘noise tolerance’ at 10) Manually count the number of clusters of segmented particles – this is recorded in the spreadsheet as the ‘number of AChR clusters’; ‘average area of AChR clusters’ and ‘fragmentation’ will be calculated automatically. Muscle Fibre Diameter Measured independently. See manuscript for methods.