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Barb Sorg’s Research Group Researching Cellular and Molecular Factors of Cocaine Addiction


Perineuronal net Intensity Program for the Standardization and Quantification of ECM Analysis

For description of protocol, see:

Please cite Slaker, Harkness, and Sorg (2016) when using the PIPSQUEAK macro. (PDF here.)

  • Slaker M, Harkness JH, and Sorg BA. A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity, IBRO Reports, 2016

You can download PIPSQUEAK from ImageJ by adding our repository to your FIJI Update Sites.


Thank you for support from:

Washington State University
WSU Postdoctoral ADARP Grant
WSU Office of Commercialization Gap Fund
Rewire Neuroscience, LLC

To install the macro:
PIPSQUEAK can be run on either FIJI or ImageJ, but to receive automatic updates to the macro, you must use FIJI and add PIPSQUEAK to your FIJI Update Sites. To do this, follow the instructions below. To use ImageJ, you will have to download and install PIPSQUEAK manually.
  • Open FIJI (or download it here: ).
  • Once FIJI is installed and opened, go to Help > Update.
  • Then, click Manage Update Sites
  • Then, click Add Update Site
  • In the center field, enter:
  • In the “Hosts” column, enter: webdav:PIPSQUEAK
  • Press enter
  • Then, click Close
  • Then, click Advanced
  • From there, you should see at the top: “PIPSQUEAK.txt
  • Click it and then click Install, and then Apply changes
  • It will install and prompt you to restart FIJI

After restarting, and go to Plugins > Macros > Install

It should have directed you to the macro folder in FIJI, where you’ll click  PIPSQUEAK.txt
Then the macro should be listed in Plugins > Macros
Every time you restart FIJI you’ll have to reinstall the macro (Plugins > Macros > Install)
If you want it to install automatically on start up, go to Plugins > Macros > Edit, then choose your “AutoRun_Scripts.imj” file. Then:
In FIJI add the lines:
run(“Install…”, “install=/Applications/”);
In FIJI add the lines:
run(“Install…”, “install=[LOCATION OF FIJI ON YOUR COMPUTER]/Macros/PIPSQUEAK.txt”);
Save, restart, and enjoy!
About the macro
New! Instructional videos:
  • Installation
  • Settings, Features, and Setup
  • Image Scale Determination
  • Single-Label Analysis
  • Double-Label Analysis
  • Triple-Label Analysis!
Change log:
0.90 CHANGE LOG:  Data log output format revised for easier export. Started logging changes
0.91 CHANGE LOG: Lowered PV pixel detection requirement. Removed Double-label Open Next Functionality. Bugs Squashed
0.92 CHANGE LOG: Splines Reticulated. Llamas Herded.
0.93 CHANGE LOG: Don’t ask about 0.93-0.942
0.943 CHANGE LOG: Lots of good things. Big changes. Re-strategized ROI seeding for double-labeling. Added user choice for ROI enlargement. Started saving ROI overlays in directory. Raw images retained for double-label double checking in semiautomated mode.
0.944 CHANGE LOG: Fix bug in saving single-label ROIs.
0.945 CHANGE LOG: Fix for double-labeled PV user check timing.
0.946 CHANGE LOG: Set default WFA ROI enlargement size to “0”. Added merged image load during double-label check steps.
0.947 CHANGE LOG: Reinstated WFA ROI expansion to prevent ROI withering during double-labeling”

>> 1.00 CHANGE LOG: Nothing changed from 0.947. Future versioning strategy will use the tenths place digit  to signify significant changes that will affect analysis results (ie 1.03 to 1.10). Data collected using versions of PIPSQUEAK that differ by a tenths place digit should not be directly compared. Data collected using versions of PIPSQUEAK similar by the tenths place digit can be compared or combined in statistical analysis (ie 1.03 and 1.04 or 1.09).

1.10 CHANGE LOG: Really cool changes! Now you can adjust the threshold of detection during your semiautomated ROI check step. It Immediately reruns ROI detection!  Save folder organization revised for double label. Now accepting different sized images! Background ROI construction revised to accept variable image dimensions. However, I addressed an issue with double-labeled cell detection in situations when the user adds additional ROIs. Therefore, results from double-label analysis using v1.10 shouldn’t be compared to previous versions. The differences between v1.10 and prior are pretty small, but ya’ know, science.”);
2.00 CHANGE LOG: Complete redesign for identification of double-labeled cells. Now, ROIs that are built during single-label identification will be maintained carried forward to the double-label step. Cells labeled by either single label are compared between labels and those with overlapping labels are identified as double labeled. This method accounts and corrects for partial overlap (removed if less that 80% overlap) and multiple overlap (two PV cells for one WFA cell). Pretty neat! One more thing, results output is improved in the Log Window. Now, each cell row contains subject, image name, and stain type information. Therefore, importing Log results into Excel is easier because you can use the “sort data” tool to help remove unwanted rows. (NOTE: Results from 2.00 and above should not be combined with earlier versions of PIPSQUEAK for statistical analysis.)
2.01 CHANGE LOG: UI improvements in cell counting function.
2.02 and 2.021 CHANGE LOG: UI improvements in settings window.
2.3 CHANGE LOG: New background selection method to improve detection of faint staining. UI improvements. Results now saved as new file each time based on date and time (no more overwriting!)
coming soon!