ImageJ Macros

The BioVoxxel Toolbox is a suite which contains plugins and some macros dealing with image filtering, image segmentation and binary image processing and analysis. The following plugins are hosted here:

• Extended Particle Analyzer
• Binary Feature Extractor
• Speckle Inspector
• Watershed Irregular Features
• EDM Binary Operations
• Filter Check
• Pseudo flat-field correction
• Convoluted Background Subtraction
• 2D Particle Distribution (Distribution_Analysis)
• Cluster Indicator
• SSIDC Cluster Indicator
• Gaussian weighted Median filter
• Adaptive Filter
• Enhance True Color Contrast
• Mode and Differential Limited Mean Binarization
• Basic Recursive Filter

Fast4DReg is a Fiji macro for drift correction for 2D and 3D video and is able to correct drift in all x-, y- and/or z-directions. Fast4DReg creates intensity projections along both axes and estimates their drift using cross-correlation based drift correction, and then translates the video frame by frame. Additionally, Fast4DReg can be used for alignment multi-channel 2D or 3D images which is particularly useful for instruments that suffer from a misalignment of channels.

This workflow describes a deep-learning based pipeline for reliable single-organoid segmentation and tracking in 2D+t high-resolution brightfield microscopy of mouse mammary epithelial organoids. The pipeline involves a four-layer U-Net to infer semantic segmentation predictions, adaptive morphological filtering to establish candidate organoid instances, and a shape-similarity-constrained, instance-segmentation-correcting tracking step to associate the corresponding organoid instances in time.

It is particularly focused on automatically detecting an organoid located approximately in the center of the first frame and track all its subsequent instances in the remaining frames, emphasizing on accurate organoid boundary delineation. Furthermore, segmentation network was trained using plausible pix2pixHD-generated bioimage data. Syntheric image simulator code and data are also available here.

It stitches 3D tiles from terabyte-size microscopy datasets. Stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly.

MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-side lightsheet illumination and dual-side camera detection.

The authors present an ImageJ-based, semi-automated phagocytosis workflow to rapidly quantitate three distinct stages during the early engulfment of opsonized beads.

The tool exports rectangular regions, defined with the NDP.view 2 software (hammatsu) from the highest resolution version of the ndpi-images and saves them as tif-files.

Click the button and select the input folder. The input folder must contain pairs of ndpi and ndpa files. The regions will be exported to a subfolder of the input folder names zones.

The tool allows to measure the area of the invading spheroïd in a 3D cell invasion assay. It can also count and measure the area of the nuclei within the spheroïd.

This tool allows to analyze morphological characteristics of complex roots. While for young roots the root system architecture can be analyzed automatically, this is often not possible for more developed roots. The tool is inspired by the Sholl analysis used in neuronal studies. The tool creates a binary mask and the Euclidean Distance Transform from the input image. It then allows to draw concentric circles around a base point and to extract measures on or within the circles. Instead of circles, which present the distance from the base point, horizontal lines can be used, which present the distance in the soil from the base-line. The following features are currently implemented:

• The area of the root per distance/depth.
• The number of border pixel per distance/depth, giving an idea of the surface in contact with the soil.
• The maximum radius per distance/depth of a root, measured at the crossing points with the circles or lines.
• The number of crossings of roots with the circles or lines.
• The maximum distance to the left and the right from the vertical axis at crossing points with the circles or lines.

Analyze the clustering behavior of nuclei in 3D images. The centers of the nuclei are detected. The nuclei are filtered by the presence of a signal in a different channel. The clustering is done with the density based algorithm DBSCAN. The nearest neighbor distances between all nuclei and those outside and inside of the clusters are calculated.

This macro toolset offers additional click tools for the rapid annotations of ROI in ImageJ/Fiji.

The ROI 1-click tools can be setup with a predefined shape, and custom actions to perform upon click (Add to ROI Manager, Run Measure, Go to next slice, run a macro command...)

To install in Fiji, just activate the ROI 1-click tools 

Blood vessels tracing in 3D image from 3D Gaussian blurring (user defined radius), local thresholding (user defined radius and offset) and 3D skeletonization. Dockerized version for BIAFLOWS,

Blood vessels tracing in 3D image from Tubeness filtering (user defined scale), 3D opening (radius set to 2), thresholding (user defined level) and 3D skeletonization.

Object tracking. For each time-frame, an image mask is obtained from median filtering (user defined radius), thresholding (user defined level) and hole filling. Convex objects are split apart by distance map watershed from regional intensity maxima (user defined noise tolerance), eroded (user defined radius) and analyzed as 3D particles (assuming some overlap between objects from a frame to the next frame). Finally, division events are analyzed and accounted for to relabel objects.

Particle tracking in 2D time-lapse based on linking closest regional intensity minima (user defined noise tolerance) detected from Laplacian of Gaussian filtered images (user defined radius). A maximum linking distance is set (user defined).

The macro will segment nuclei and separate clustered nuclei in a 3D image using a 2D Gaussian blur, followed by Thresholding, 2D hole filling and a 2D watershed. As a result an index-mask image is written for each input image.

3D spot detection using the Determinant of Hessian (DoH) and the detection of 3D minima.

This workflow detects spots in a 2D image by filtering the image by Laplacian of Gaussian (user defined radius), thresholding (user defined threshold) and finding local intensity maxima in mask distance map (Dmap).

OligoMacro Toolset, is an ImageJ macro-toolset aimed at isolating oligodendrocytes from wide-field images, tracking isolated cells, characterizing processes morphology along time, outputting numerical data and plotting them. It takes benefit of ImageJ built-in functions to process images and extract data, and relies on the R software in order to generate graphs.

We have developed WormScan, an automated image acquisition system that allows quantitative analysis of each of these four phenotypes on standard NGM plates seeded with E. coli. This system is very easy to implement and has the capacity to be used in high-throughput analysis.

Protein array is used to analyze protein expressions by screening simultaneously several protein-molecule interactions such as protein-protein and protein-DNA interactions. In most cases, the detection of interactions leads to an image containing numerous lines of spots that will be analyzed by comparing tables of intensity values. To describe the observed different patterns of expression, users generally show histograms with the original associated images [1]. The “Protein Array Analyzer” gives a friendly way to exploit this type of analysis, thus allowing quantification, image modeling and comparative analysis of patterns.

The Protein Array Analyzer, which was programmed in ImageJ’s macro language, is an extention of the Dot Blot Analyzer, [2], [3] a graphically interfaced tool that greatly simplifying analysis of dot arrays.

This workflow detects spots from a 3D image by using straightforward set of ImageJ components. It receives the Laplacian Radius and the Threshold  value s input.

This workflow detects spots in a 2D image by filtering the image by Laplacian of Gaussian (user defined radius) and detecting regional intensity minima (user defined noise tolerance).

The macro will segment nuclei and separate clustered nuclei in a 3D image using a distance transform watershed. As a result an index-mask image is written for each input image.

The macro will segment nuclei and separate clustered nuclei using a binary watershed. As a result an index-mask image is written for each input image.

CLIJ2 is a GPU-accelerated image processing library for ImageJ/Fiji, Icy, Matlab and Java. It comes with hundreds of operations for filtering, binarizing, labeling, measuring in images, projections, transformations and mathematical operations for images. While most of these are classical image processing operations, CLIJ2 also allows performing operations on matrices potentially representing neighborhood relationships between cells and pixels.

CLIJ2 was developed to process images from fluorescence microscopy data of developing cells, tissues, organoids and organisms.

InspectJ is a free ImageJ/FIJI tool to inspect digital image integrity.

InspectJ_v2 is a newer version for advanced users. It applies additional features like histogram equalization and gamma correction for improved image inspections.

This is a classical workflow for spot detection or blob like structures (vesicules, melanosomes,...)

Step 1 Laplacian of Gaussian to enhance spots . Paraeters= radius, about the average spot radius

Step 2 Detect minima (using Find Maxima with light background option to get minima). Parameter : Tolerance to Noise: to be tested, hard to predict. About the height of the enhanced feautures peaks

This script includes a rough feature detection and then fine 2D Gaussian algorithm to fit Gaussians within detected regions. This macro is unique because the ImageJ/Fiji curve fitting API only supports 1-D curve. I get around this by linearising the equation. This implementation is for isotropic (spherical) or anistropic (longer in x/y) diagonally covariant Gaussians but not fully covariant Gaussians (anisotropic and rotated). 

HyphaTracker propose a workflow for time-resolved analysis of conidia germination. Each part of this workflow can also be used independnatly , as a toolbox. It has been tested on bright-field microscopic images of conidial germination. Its purpose is mainly to identify the germlings and to remove crossing hyphae, and measure the dynamics of their growth.

The purpose of the workflow is ....

First you need

"The plugin analyzes fluorescence microscopy images of neurites and nuclei of dissociated cultured neurons. Given user-defined thresholds, the plugin counts neuronal nuclei, and traces and measures neurite length."[...]" NeuriteTracer is a fast simple-to-use ImageJ plugin for the analysis of outgrowth in two-dimensional fluorescence microscopy images of neuronal cultures. The plugin performed well on images from three different types of neurons with distinct morphologies."

This plugin requires parameter setting: Threshold levels and scale (see more details on the related publication)

The skin tools measure the thickness of the epidermis and the interdigitation index.

The input images are masks that represent the epidermis and that have been created from images of stained histological sections. The mask must touch the left and right border of the image. The dermal-epidermal border must be on the lower site of the image. The interdigitation index can be measured for one or more segments per image. As a measure of the thickness of the epidermis the lengths of a number of random line segments are measured. The line segments start at the lower border, are perpendicular to the lower border and end at the opposite border of the mask.

See installation Instructions on the website.

The Adipocytes Tools help to analyze fat cells in images from histological section. This is a rather general cell segmentation approach. It can be adapted to different situations via the parameters. This means that you have to find the right parameters for your application.

Sample Image: [0178_x5_3.tif](http://dev.mri.cnrs.fr/attachments/190/0178_x5_3.tif)

Neural Circuit Tracer (NCTracer) is open source software for automated and manual tracing of neurites from light microscopy stacks of images. NCTracer has more than one workflow available for neuron tracing. 

"The Neural Circuit Tracer is open source software built using Java (Sun Microsystems) and Matlab (MathWorks, Inc., Natick MA). It is based on the core of ImageJ (http://rsbweb.nih.gov/ij) and the graphic user interface has been developed by using Java Swings. The software combines anumber of functionalities of ImageJ with several newly developed functions for automated and manual tracing of neurites. The Neural Circuit Tracer is designed in a way
that will allow the users to add any plug-ins developed for ImageJ. More importantly, functions written in MatLab and converted into Java with Matlab JA toolbox can also be added to the Neural Circuit Tracer." 

This ImageJ plug-in is a compilation of co-localization tools. It allows:

-Calculating a set of commonly used co-localization indicators:

Pearson's coefficient Overlap coefficient k1 & k2 coefficients Manders' coefficient Generating commonly used visualizations:

-Cytofluorogram

Having access to more recently published methods:

-Costes' automatic threshold

Li's ICA Costes' randomization Objects based methods (2 methods: distances between centres and centre-particle coincidence)

Bio Image Analysis tool from REF

ImageJ macro for the morphometry of neurites. > NeurphologyJ; it is capable of automatically quantifying neuronal morphology such as soma number and size, neurite length, neurite ending points and attachment points. NeurphologyJ is implemented as a plugin to ImageJ, an open-source Java-based image-processing and analysis platform.

Estimate the frequency of hepatitis C virus infected cells based on the intensity of viral antigen associated immunofluorescence. 

The core is an ImageJ Macro, so it's easy to modify for one's own needs (Link to the code). 

In this workflow, you can use MorphoLibJ to generate accurate morphometric measurements

• First the fibers are segmented by mathematical morphology:
  • for example by using MorphoLibJ:
    • Create a marker image by creating a rough mask with extended regional maxima (similar to Find Max), such that you have one max per fiber
    • Use the marker controlled watershed (in MorpholLibJ/ Segmentation/ marker controlled watershed) : indicate the original grayscale image as the input, Marker will be your maxima image, select None for mask
    • it will create a label mask of your fibers
•  In MorphoLibJ /analyze/ select Region Morphometry: this will compute different shape factors which are more robust than the ones implemented by default in ImageJ
• Export the result table created to a csv file
• Then for example in Matlab or R, you can apply a PCA analysis (Principal component analysis) followed by a k-means with the number of class (clusters) (different fibers type) you want to separate.
• You can then add this class as a new feature to your csv file.
• From this you can sort your labelled fibers into these clusters for a visual feedback or further spatial analysis

Quote:

The "Angiogenesis Analyzer" allows analysis of cellular networks. Typically, it can detect and analyze the pseudo vascular organization of endothelial cells cultured in gel medium

...a simple tool to quantify the ETFA (Endothelial Tube Formation Assay) experiment images by extracting characteristic information of the network.

The outputs are network feature parameters.

Sample images

http://image.bio.methods.free.fr/ij/ijmacro/Angiogenesis/HUVEC-Pseudo-Phase-Contrast.tif.zip

http://image.bio.methods.free.fr/ij/ijmacro/Angiogenesis/HUVEC-Fluo.tif.zip

Source code

https://imagej.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt

Evaluates the orientation of fiber orientation pattern and plots the results in the image. It calculates gradient in x and y direction. - then calculates the eigenvector of nematic tensor, which is the orientation of the pattern.

The workflow consists of firstly identifying spot (which can be also gravity center of cells identified by another method), and then secondly compute trajectories by linking these spots by global optimisation with a cost function. This method is part of the methods evaluated in Chanouard et al (2014) as "method 9" and is described in detail in its supplementary PDF (page 65).

Dependencies

Following plugins are required.

1. JAR to be placed under IJ plugin directory
2. A pdf file with instructions and output description is also available in the zip .
3. MTrackJ : Used for visualization of tracks. Preinstalled in Fiji.
4. Imagescience.jar: This library is used by MTrackJ. Use update site to install this plugin.
5. jama.jar. Preinstalled in Fiji.

##Advantages:

• support blinking (with a parameters allowing it or not)
• fast,
• can be used in batch, some analysis results provided.
• No dynamic model.
• The tracking part is not dependent of ImageJ.

Pitfalls:

• does not support division
• the optimization algorithm used is a simulated annealing, so results can be slightly different between two runs.
• No Dynamic model (so less good results but can be used for a first study of the kind of movements)

##The sample data

The parameters used for this example data Beads, were

1. detection: 150
2. the max distance in pixels: 20
3. max allowed disappearance in frame: 1

The root tools help to efficiently measure the following characteristics of plant roots: the angle of the opening of the whole root the depth to which it goes down the number of roots at multiple depths (for example 30cm, 35cm, ...) the diameters of the roots at multiple depths (for example 30cm, 35cm, ...)