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Analyzes, processes and visualizes multi-dimensional microscopy images. BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes.


An open source application for the visualization and analysis of 4D biological datasets. Developed by researchers, it is primarily used for the analysis and quantification of 4D live-imaged confocal data. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.


A statistical method for tracing the branch centerlines of neurons based on Bayesian multi-object tracking using probability hypothesis density (PHD) filtering. hgpush is able to simultaneously trace out multiple neuron structures in a probabilistic fashion so that the same neuron segments may be covered multiple times and are thus supported by more evidence. The main novelty is that it combines the problems of neuron segment detection and linking into one framework by performing simultaneous multi-object tracking. hgpush solves the computational problems of direct Bayesian multi-object tracking and allows convenient handling of bifurcations and terminations during the tracing process by modeling of spawned objects and observation clutter.

3D Tissue Organization Toolbox

Provides all the necessary methods for nuclei segmentation, cell identification and analysis of cell interaction in 3D. 3D Tissue Organization Toolbox is an ImageJ plugin that covers different aspects of spatial analysis of tissues in 3D, ranging from nuclei segmentation to analysis of different aspects of cellular interaction and network mapping. It was used to investigate current and novel aspects of the unique architecture of the pancreatic islet of Langerhans.


A software application on MATLAB to visualize surface data from 3D fluorescence microscopy. Map3-2D accurately accurately projects up to five-dimensional (5D) fluorescence microscopy image data onto full-content 2D maps. Similar to the Earth's projection onto cartographic maps, Map3-2D unfolds surface information from a stack of images onto a single, structurally interconnected map. By cross-referencing between the 2D map and the original image stack, precise quantitative analyses of intensities and shapes are easily and intuitively executable.


Calculates the fractal dimensionality of a 3D structure. calcFD is designed to work with intermediate files from FreeSurfer analysis pipeline, but can also use other volumes. The toolbox includes options to use different masking files and is implemented to use either the box-counting or dilation algorithms and to use either the filled volume or just the surface of the structure. The toolbox can easily be run on all of the participants in a FreeSurfer subject folder, or just on specified subject folders. The Matlab toolbox also includes several functions designed to improve functionality, such as the automatic ‘cropping’ of the volume space to the smallest bounding box necessary to contain the volume, improving computation time drastically. Example files are also provided to aid in using the toolbox for the user‘s needs.

APP / All-Path-Pruning

A software for 3D neuron tracing. APP2 prunes an initial reconstruction tree of a neuron’s morphology using a long-segment-first hierarchical procedure instead of the original termini-first-search process in APP. This method allows users to trace large images. APP2 was bench-tested on 700 3D microscopic images and it was found that APP2 can generate more satisfactory results in most cases than several previous methods. This software has been implemented as an open-source Vaa3D plugin.


Proposes a large collection of generic tools based on mathematical morphology to process binary and grey-level 2D and 3D images, integrated into user-friendly plugins. The library provides different categories of functions, corresponding to standard image processing workflows: (i) image processing and filtering; (ii) segmentation; (iii) post-processing; (iv) quantitative analysis; (v) library re-usability. The cell-resolved data provided by MorphoLibJ will be useful for the analysis of cell lineage, and the modelling of plant growth and morphogenesis in 3D.

KiT / Kinetochore Tracking

An easy-to-use, open-source software package for tracking kinetochores from live-cell fluorescent movies. KiT supports 2D, 3D and multi-colour movies, quantification of fluorescence, integrated deconvolution, parallel execution and multiple algorithms for particle localization. KiT includes a user-friendly GUI (Graphical User Interface) for selecting ROIs (Regions Of Interest; to select cells and exclude spurious background fluorescence), parameter configuration and execution of KiT. Tracking may be executed from within the GUI or later. The GUI allows selection of particle detection algorithms per channel and modification of the most commonly used options. KiT also includes a GUI for post-tracking processing, enabling basic diagnostics and track quantification, such as kinetochore speeds and autocorrelation. The GUI collates data from each tracked ROI and saves a .mat file for later user-specific processing.

Spectral Imaging Toolbox

Measures accurately cell membrane lipid packing without cytosolic contributions using a single dye. The Spectral Imaging Toolbox was designed for spectral analysis of high magnification images of single or sub-confluent cells, vesicles and microbubbles. This method permits to process and analyze a dataset containing over 1500 cells in a few hours. It also addresses a growing need to process large spectral imaging datasets and data from experiments with multiple exposures.

Rayburst sampling

An algorithm for automated three-dimensional shape analysis from laser scanning microscopy images. The Rayburst algorithm provides a primitive for the development of higher level algorithms that solve specific shape analysis problems. Examples are provided of applications to 3D neuronal morphometry: (i) estimation of diameters in tubular neuronal dendritic branching structures, and (ii) measurement of volumes and surface areas for dendritic spines and spatially complex histopathologic structures.


A pipeline for the reconstruction of discontinuous neuronal morphology in noisy images. SparseTracer is based on two methods. One is the region-to-region connection (RRC) method for detecting the initial part of a neurite, which can effectively gather local cues, i.e., avoid the whole image analysis, and thus boosts the efficacy of computation. The other is constrained principal curves method for completing the neurite reconstruction, which uses the past reconstruction information of a neurite for current reconstruction and thus can be suitable for tracing discontinuous neurites. SparseTracer is able to deal with the large-scale image dataset.

Neuron Crawler

An automatic tracing algorithm. Neuron Crawler works by first tracing a region of interest (e.g., around the soma), and then iteratively tracing in adjacent image tiles to grow the neuron structure in 3D to its termination point within the image. It makes it possible to reconstruct very large 3D images. Experimental results show that Neuron Crawler can achieve reconstruction accuracy that is comparable to several state-of-the art algorithms, but with much less computational cost. Neuron Crawler is implemented as a plugin of Vaa3D.


An automatic tracing framework for neuron reconstruction that does not require substantial human intervention. SmartTracing algorithm has been implemented as a plugin of Vaa3D, which is the common platform to implement algorithms for the BigNeuron project bench-testing. SmartTracing does not require human input of training exemplars and can self-adapt to different types of neuroimage data. Additionally, the method can be applied to improve the performance of other existing tracing methods.


A modular software tool for efficient quantification of biological tissues based on volume data obtained by biomedical image modalities. TiQuant includes a number of versatile image and volume processing chains tailored to the analysis of different tissue types which have been experimentally verified. It implements a novel method for the reconstruction of three-dimensional surfaces of biological systems, data that often cannot be obtained experimentally but which is of utmost importance for tissue modelling in systems biology.


Analyses raw confocal microscopy images. Analyses options include the whole image, specific regions of the image (cropping) and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either re-orientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters.


An interactive 3D axon tracking and labeling tool to obtain quantitative information by reconstruction of the axonal structures in the entire innervation field. AxonTracker-3D has been developed to facilitate the connectome function analysis in large-scale quantitative neurobiology studies. It can display the three orthogonal views of the current location of the centerline along with a visualization of the tracking results. The workflow consists of three steps: (i) re-slice the axon tubes along its orientation; (ii) extract 2D and 3D features from the slices and spheres rounding the center points; (iii) select samples to train AdaBoost classifier. Questions such as whether the spatial distribution of the axons are random in nature or follow a certain pattern can be answered with this tool.

Simple Neurite Tracer

An application for semi-automated tracing of neurons to quickly annotate noisy datasets and construct complex neuronal topologies. Simple Neurite Tracer is designed to allow easy semi-automatic tracing of neurons or other tube-like structures (e.g. blood vessels) through 3D image stacks. The plugin has built-in tools for analysis and hardware accelerated 3D visualization of the results. Data can be imported and exported in SWC files for interaction with other software, or details of the traces can be exported as CSV files for analysis in spreadsheets or statistical software. The native file format is open and XML-based.

iRoCS Toolbox

An interactive Qt4 analysis tool for attaching the intrinsic root coordinate system to 3-D confocal recordings of the Arabidopsis root apical meristem. This allows quantitative comparative studies of root populations in a sound and intuitive context. iRoCS Toolbox consists of the data browser labelling and a set of command line tools for batch processing. Main features are: (i) 3-D Visualization of the volume in an orthographic view; (ii) manual annotation of anatomical reference structures (QC, nuclei); (iii) automatic detection and annotation of cells and/or nuclei with several meaningful properties, like position, radius, tissue, cell file, cell cycle state, volume; (iv) full control. Any step can be manually corrected and re-fed into the modeling; (v) retrieval and localization of key events like cell divisions.


A plugin for bone image analysis in ImageJ. BoneJ provides free, open source tools for trabecular geometry and whole bone shape analysis. It calculates several trabecular, cross-sectional and particulate parameters in a convenient format. Java technology allows BoneJ to run on commodity computers, independent of scanner devices, fully utilising hardware resources. ImageJ’s plugin infrastructure provides a flexible working environment that can be tailored to diverse experimental setups. BoneJ is a working program and a starting point for further development, which will be directed by users’ requests and the emergence of new techniques.


Builds 3D neural meshes. Neuronize is a set of methods designed taking the morphological information extracted through computer-aided tracing applications as the starting point. The software generates 3D models for neuronal cells that approximate the cell membrane at different resolution levels, allowing balance to be reached between the complexity and the quality of the final model. The software is conceived to overcome the problems caused by the unrealistic somata and low quality unconnected 3D mesh.

Open Snake Tracing System

A broadly applicable algorithm and a comprehensive open-source software implementation for automated tracing of neuronal structures in 3-D microscopy images. Open Snake Tracing System has been integrated to the Farsight toolkit. The core 3-D neuron tracing algorithm is based on three-dimensional (3-D) open-curve active Contour (Snake). It is initiated from a set of automatically detected seed points. A suite of pre-processing algorithms enable the system to accommodate diverse neuronal image datasets by reducing them to a common image format. These algorithms form the basis for a comprehensive, scalable, and efficient software system developed for confocal or brightfield images. It provides multiple automated tracing modes. The user can optionally interact with the tracing system using multiple view visualization, and exercise full control to ensure a high quality reconstruction.