‘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.