Table 2.

Sources and Solutions to Potential Errors

Problem	Possible cause	Solution
Macro started and immediately shows the message “Macro finished.” Without processing	Wrong input folder	Use the log file to check which input folder was selected (also, check that the correct files are in the input folder—i.e., if there are no .tiff files in it, it generally does not work).
Macro error “Stack required in line XXX […]”	MIPs selected as input instead of 3D stacks	Click okay and double‐check that the selected input folder contains the correct 3D stacks instead of MIPs (which are always indicated with “MAX_” in their name)
Memory issues		Edit › Options › Memory & Threads > change memory > restart Fiji
Step1_cziToTiffTool: “Stack is larger than 4 GB. Most TIFF readers will only open the first image. Use this information to open as raw:[…]”		Normally does not require any intervention
The output MIP folder is empty	The tool was probably run on MIPs instead of 3D stacks	Double‐check that the correct input folder was selected
Step2_DeconvolutionTool: Unrecognized command “DeconvolutionLab2 Run” in line 281	Missing plugin	Go to http://bigwww.epfl.ch/deconvolution/deconvolutionlab2/ and follow the instructions for installation
Step2_DeconvolutionTool: Unrecognized command “Diffraction PSF 3D” in line 215	Missing plugin	Go to https://www.optinav.info/Iterative‐Deconvolve‐3D.htm or https://imagej.net/Diffraction_PSF_3D and follow instructions for installation
Step2_DeconvolutionTool: Image “Final Display of RL” not found	Macro continuing to next step without finishing deconvolution	This usually happens if the images are large. Two options to solve it: 1) Increase wait time in line 282 (e.g., wait(80000)) 2) Make images smaller before running the deconvolutionTool, using the subregionTool (recommended)
Step4_subregionTool: Java.io.FileNotFoundException: FilePath\RoiSetLine.zip (the system cannot find the file specified).	RoiSetLine.zip not found.	1) RoiSetLine.zip does not exist > needs to be created by the user 2) RoiSetLine.zip exists but not in the correct folder > make sure the file is in the same folder as the input data 3) RoiSetLine.zip name is wrong > double‐check that it is correctly named/spelled (case‐sensitive!)
Step8_quantificationTool: Unrecognized command "3D Manager Options" line 61	3D ImageJ Suite Plugin missing	See Basic Protocol 1 on how to update sites
Step8_quantificationTool: Unrecognized command "Summarize Skeleton"	Neuroanatomy plugin missing	See Basic Protocol 1 on how to update sites