PIRATE

PIRATE is an ImageJ/Fiji plugin for quantitative fluorescence intensity standardization in biological fluorescence imaging. It provides an easy-to-use graphical interface for intensity normalization of single images and image stacks, supporting Max, Min-Max, Percentile, and PIRATE normalization workflows.

PIRATE is designed to improve the consistency and comparability of fluorescence intensity distributions across images, especially in workflows where intensity variation, background fluctuation, or photon-limited acquisition conditions affect downstream visualization and quantitative analysis.

Preview

Graphical User Interface

Example Output

Raw image	PIRATE output

Features

• ImageJ/Fiji plugin with a Swing-based graphical user interface.
• PIRATE adaptive patch-based fluorescence intensity normalization.
• Baseline normalization modes, including:
  • Max normalization
  • Min-Max normalization
  • Percentile normalization
  • PIRATE
• Support for 8-bit, 16-bit, and 32-bit grayscale images.
• Support for both single images and image stacks.
• Histogram visualization for single-image normalization.

Installation

Option 1: Use the bundled JAR

1. Copy pirate_1.0.0.jar into the plugins/ folder of Fiji or ImageJ.
2. Restart Fiji/ImageJ.
3. Open an image.
4. Run the plugin from:

Plugins > PIRATE > PIRATE

Option 2: Download a release JAR

1. Download pirate_1.0.0.jar from the GitHub Releases page.
2. Copy the JAR file into the plugins/ folder of Fiji or ImageJ.
3. Restart Fiji/ImageJ.
4. Open an image and run:

Plugins > PIRATE > PIRATE

Option 3: Build from source

Requirements:

• Java 8 or later
• Maven 3.x
• Fiji or ImageJ

Build the project with:

mvn clean package

After building, the plugin JAR will be generated under:

target/pirate_1.0.0.jar

Copy this JAR file into the Fiji/ImageJ plugins/ folder, then restart Fiji/ImageJ.

Usage

1. Open an 8-bit, 16-bit, or 32-bit grayscale image or image stack in Fiji/ImageJ.
2. Run:

Plugins > PIRATE > PIRATE

3. Select a normalization mode:
   • PIRATE: adaptive patch-based fluorescence intensity normalization.
   • Percentile normalization: normalization based on user-defined lower and upper percentiles.
   • Min-Max normalization: normalization based on the occupied intensity range.
   • Max normalization: normalization by the maximum occupied intensity.
4. Choose whether to display the histogram.
5. Click START PROCESS.

For image stacks, PIRATE estimates normalization parameters from the first slice and applies the resulting normalization model to all slices in the stack.

Normalization Modes

PIRATE normalization

The PIRATE mode performs adaptive patch-based fluorescence intensity normalization. It is designed for biological fluorescence images where local intensity distributions may vary because of uneven illumination, background fluctuation, photon-limited acquisition, or sample-dependent signal heterogeneity.

Percentile normalization

Percentile normalization rescales image intensity using user-defined lower and upper percentile thresholds. This mode is useful when extreme intensity values should be excluded from the normalization range.

Min-Max normalization

Min-Max normalization rescales the occupied intensity range of the image to the output dynamic range.

Max normalization

Max normalization rescales image intensity according to the maximum occupied intensity value.

Repository Layout

.
|-- pirate_1.0.0.jar
|-- pom.xml
|-- src/main/java/org/srwiki/pirate/PIRATE_Normalization.java
|-- src/main/resources/plugins.config
|-- src/main/resources/pirate_logo_3x4_transparent.png
|-- src/main/resources/1.png
`-- examples/
    |-- GUI.png
    |-- RAW.png
    `-- PIRATE_output.png