GDSC Single Molecule Light Microscopy (SMLM) ImageJ Plugins

The GDSC Single Molecule Light Microscopy (SMLM) plugins provide various tools for single molecule localisation analysis. This includes PALM, STORM and other single molecule microscopy methods.

The plugins provide tools to:

• Fit an image, or series of images, using a 2D Guassian Point Spread Function (PSF)
• Save results to a table, a file, an image and/or to memory
• Trace or cluster localisations through time to identify molecules
• Correct drift in long time course images
• Estimate fluorophore dark-time and blinking rate
• Create localisation density images
• Create custom PSFs from calibration images
• Create simulated data using a Gaussian or custom PSF with configurable molecule populations and diffusion
• Calibrate the gain and read noise of your microscope camera
• Estimate noise in an image
• Estimate resolution using Fourier Ring Correlation

Results can be loaded from file for analysis using the following formats:

• SMLM Text/Binary
• RapidSTORM
• Nikon DSTORM

The code is split into modules:

• gdsc-smlm: Contains utilities for SMLM data analysis
• gdsc-smlm-ij: Contains SMLM plugins for ImageJ

Citation

GDSC SMLM: Single-molecule localisation microscopy software for ImageJ [version 1; peer review: 1 approved].
Etheridge TJ, Carr AM and Herbert AD.
Wellcome Open Res 2022, 7:241

Documentation

See the latest documentation on ReadTheDocs.

Install

The SMLM plugins are distributed using an ImageJ2/Fiji update site.

To install the plugins using Fiji (an ImageJ distribution) just follow the instructions How_to_follow_a_3rd_party_update_site and add the GDSC SMLM update site. All the plugins will appear under the 'Plugins > GDSC SMLM' menu.

Installation from source

The source code is accessed using git and built using Maven.

The code depends on the gdsc-test, gdsc-ij-parent and gdsc-core artifacts so you will have to install these to your local Maven repository before building:

1. Clone the required repositories

    git clone https://github.com/aherbert/gdsc-test.git
    git clone https://github.com/aherbert/gdsc-ij-parent.git
    git clone https://github.com/aherbert/gdsc-core.git
    git clone https://github.com/aherbert/gdsc-smlm.git
   

2. Build the code and install using Maven

    cd ../gdsc-test
    mvn install
    cd ../gdsc-ij-parent
    mvn install
    cd ../gdsc-core
    mvn install
    cd ../gdsc-smlm
    mvn package
   

   This will produce a gdsc-smlm-ij_-[VERSION].jar file in the target directory of the gdsc-smlm-ij module. Dependencies can be copied into the target/dependencies directory using:

    mvn dependency:copy-dependencies
   

3. Installation into a Fiji/ImageJ2 install can be performed using the scijava maven goal to populate the application:

    cd gdsc-smlm-ij
    mvn scijava:populate-app -Dscijava.app.directory=/usr/local/fiji
    cd ..
   

   where /usr/local/fiji is the root directory of the ImageJ install.

4. Manual installation must copy the gdsc-smlm-ij_* jar into the plugins directory of ImageJ.

   Copy the dependencies into the plugins directory (or onto the Java classpath). Note that the Maven package routine puts all dependencies into the target/dependencies directory even if they are not required by the SMLM code (it does not check what functions are actually used by the code). The libraries required are:

    gdsc-core
    gdsc-core-ij
    gdsc-smlm
    commons-math3
    commons-lang3
    commons-rng-client-api
    commons-rng-core
    commons-rng-simple
    commons-rng-sampling
    ejml
    fastutil-core
    guava
    JTransforms
    JLargeArrays
    mfl-core
    mxparser
    protobuf-java
    protobuf-java-util
    quickhull3d
    xstream
    xpp3_min
   

   This excludes the 3D_Viewer functionality. View the dependencies using:

    mvn dependency:tree
   

5. The plugins will now appear under the 'Plugins > GDSC SMLM' menu in ImageJ.

Running from source

Maven can be used to run ImageJ using a profile defined in the gdsc-ij-parent POM. This profile compiles all classes and then executes ImageJ with the appropriate Java classpath.

To run the GDSC SMLM plugins requires that they are detected by ImageJ. The plugins use the ImageJ v1 plugin architecture which uses a search in the plugins directory for classes with an underscore in the name. To avoid having to package the classes to a jar file a bootstrap GDSC plugin loader is provided. To run the GDSC SMLM plugins requires creating a link from the compilation target folder to a plugins directory:

    cd gdsc-smlm-ij
    ln -s target/classes plugins
    mvn -P run-imagej

On start-up ImageJ will search in the plugins directory and find the bootstrap plugin. This can then be run from the ImageJ Plugins menu to load the plugins defined in the project's ImageJ plugins.config file.

Note: This file is normally detected by ImageJ when loading plugin jar files to identify the available plugins. The bootstrap plugin has been written to duplicate this functionality by reading the configuration and populating the ImageJ menu. It allows ImageJ to be run with the latest classes using only the compile goal and avoids having to package the classes to a jar. An alternative is to package the class files and create a link from the jar location to a plugins directory:

    cd gdsc-smlm-ij
    ln -s target plugins
    mvn compile jar:jar
    mvn -P run-imagej

Note that when using this approach ImageJ may detect duplicate plugins if the sources jar file has previously been created using the package goal. This can be avoided by cleaning the target directory using mvn clean.

Using the jar approach will replicate the ImageJ Plugins > GDSC SMLM menu structure.

Modifying the source

The gdsc-smlm code was developed using the Eclipse IDE.

Details of how to open the source code with Eclipse can be found in the eclipse folder.

Legal

See LICENSE

About

Owner(s)

Alex Herbert

Institution

Genome Damage and Stability Centre, University of Sussex

URL

GDSC SMLM ImageJ plugins