Quantifying synapses with Puncta Analyzer plugin

Dear all,

I need to analyse some fluorescent confocal images stained with double presynaptic markers and other images stained with pre- and postsynaptic markers. In order to do so, I was told to try the ImageJ plugin “Puncta Analyzer”. However, I’m experiencing difficulties with it, and these are the same that a coupled of years ago started a discussion in ImageJ’s mailing list http://imagej.1557.x6.nabble.com/Puncta-Analyzer-td4892020.html

I’ve managed to install Puncta-Analyzer v1.1 in Fiji-ImageJ (ImageJ v1.51a; Java 1.8.0_66), but couldn’t get it to show nor save any results. I then ran the plugin in an older version of ImageJ (1.46r; Java 1.8.0_66), only to obtain the same exception:

java.lang.NoSuchMethodError: ij.ImagePlus.getMask()[I
	at Puncta_Analyzer$ScopeImageUtils.contractHistogram(Puncta_Analyzer.java:708)
	at Puncta_Analyzer.locatePunctaInColorBand(Puncta_Analyzer.java:660)
	at Puncta_Analyzer.locatePuncta(Puncta_Analyzer.java:620)
	at Puncta_Analyzer.run(Puncta_Analyzer.java:240)
	at ij.plugin.filter.PlugInFilterRunner.processOneImage(PlugInFilterRunner.java:256)
	at ij.plugin.filter.PlugInFilterRunner.<init>(PlugInFilterRunner.java:105)
	at ij.IJ.runUserPlugIn(IJ.java:187)
	at ij.IJ.runUserPlugIn(IJ.java:196)
	at ij.IJ.runPlugIn(IJ.java:152)
	at ij.Executer.runCommand(Executer.java:127)
	at ij.Executer.run(Executer.java:64)
	at java.lang.Thread.run(Thread.java:745)

Since in the discussion I mentioned, the possibility of fixing this issue with Punta Analyzer was considered, maybe by developing a Puncta-Analyzer v2, I was wondering whether something was advanced in this sense. I believe that, although quite some time has already passed since then, @ctrueden may know something about it.

Thank you all in advance.

Best regards,
Carlos Pretell Annan

As described in the thread there seems to be a newer version available. Have you tried the 2.0 version:

Dear Bio7,

Firstly, thank you very much for your answer. I’ve already tried downloading Puncta-Analyzer v2.0 from the github link you suggested. However, I couldn’t manage to install it. Here are the instructions they provide, but, for instance, I can’t find neither the “‘mvn package’ in project directory” nor the ‘.jar file from target/ to ImageJ’s plugins/ directory’.

Build Instructions:

Install JDK

Install maven

Fork the puncta analyzer repo (or download project zip)

Execute ‘mvn package’ in project directory (where the pom.xml is located)

Copy the .jar file from target/ to ImageJ’s plugins/ directory

I was wondering whether the 2.0 version is actually available.

Thank you very much in advance.


You have to install maven and follow the instructions to built the plugin.

However I would contact the author if he could distribute a binary for a simple download which you can put in the plugins directory.

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If you have trouble with installing this, you could also try the Example Speckle pipeline for CellProfiler: http://cellprofiler.org/examples/#Speckles

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What error message do you get when running mvn package from the command line?

When I tried after cloning the repository, running mvn from the command line successfully built the following jar file (unzip the file to get the jar): puncta_analyzer-2.0.0-SNAPSHOT.zip (24.4 KB)

If you don’t succeed building it yourself, just take that file and unzip it into your ./plugins/ folder. That should allow you trying the plugin.

From your description, I’m not sure though if the Puncta Analyzer plugin will be optimal for your use case. If you also post some example images, someone here on the forum might have better suggestions how to analyze them.


Dear @imagejan,

thank you very much for the file, I can now run Puncta Analyzer with Fiji.

Here I have an example of the kind of images I need to analise: it’s a two channels RGB image of mouse brain cortex stained with a presynaptic and a postsynaptic markers, from which I’d like to know the number of putative synapses by means of puncta counting.

Thank you all very much for your help.

dear Pretell,
I am also trying to analyse some fluorescent confocal images stained with double presynaptic markers and other images stained with pre- and postsynaptic markers. I tried to download the puncta analyzer plugin from the file provided in the comment by ImageJan. Did this work for you? I am able to download the file, but it does not appear on the plugin menu in imagej. Any advice?
Thank you!

Dear @jroma,

I’m glad to know that this topic is still active after so much time. However, I haven’t continued with that analysis, so I didn’t use this plugin that much.

Regarding the issue you are experiencing, have you tried installing the plugin? Once you open FIJI-ImageJ, you can go to ‘Plugins’, then ‘Install…’ and select the file puncta_analyzer-2.0.0-SNAPSHOT.jar in the .zip file. Another way would be to unzip that file into fiji’s ‘/plugins/’ folder. I hope that works fine!

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I’m trying to find the best way to quantify pre- and post-synapse in brain tissue.

Firstly, I tried to use ImageJ plugin, Puncta Analyzer.

I installed java JDK and maven by github description.

But, I failed mvn package usage…

Secondly, I found same problem with me and followed by.

I move jar files to ImageJ Plugin folder.


However, I couldn’t find puncta analyzer in ImageJ screen.

How can I solve this problem…?

Or please recommend me another synapse quantification program.