Filament tracing

Junction Mapper is a semi-automated software (Java Desktop application) for analysing data from images of cells in close proximity to each other in monolayers. The focus of Junction Mapper is to measure the morphology of cell boundaries, define single junctions and quantify the length, area and intensity of the staining of different proteins localised at cell-cell contacts. The output are various unique parameters that assess the contacting interface between cells and up to two junctional markers.

ClearMap is a toolbox for the analysis and registration of volumetric data from cleared tissues.

It was initially developed to map brain activity at cellular resolution in whole mouse brains using immediate early gene expression. It has since then been extended as a tool for the qunatification of whole mouse brain vascualtur networks at capilary resolution.

It is composed of sevral specialized modules or scripts: tubemap, cellmap, WobblyStitcher.

ClearMap has been designed to analyze O(TB) 3d datasets obtained via light sheet microscopy from iDISCO+ cleared tissue samples immunolabeled for proteins. The ClearMap tools may also be useful for data obtained with other types of microscopes, types of markers, clearing techniques, as well as other species, organs, or samples.

Vaa3d BJUT Fast Marching Spanning Tree algorithm dockerised workflow for BIAFLOWS

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.

3D Neuron Tracing with a Dockerized version of Vaa3D MOST Raytracer.

3D Neuron Tracing using Dockerized version of Vaa3D Minimum Spanning Tree (MST).

Rivuletpy dockerised workflow for BIAFLOWS.

Vaa3d All-Path-Pruning 2.0 (APP2) dockerised workflow for BIAFLOWS.

Fiji plugin for detecting, tracking and quantifying filopodia

Paintera is a general visualization tool for 3D volumetric data and proof-reading in segmentation/reconstruction with a primary focus on neuron reconstruction from electron micrographs in connectomics. It features/supports:

•  Views of orthogonal 2D cross-sections of the data at arbitrary angles and zoom levels
•  Mipmaps for efficient display of arbitrarily large data at arbitrary scale levels
•  Label data
  •  Painting
  •  Manual agglomeration
  •  3D visualization as polygon meshes
    •  Meshes for each mipmap level
    •  Mesh generation on-the-fly via marching cubes to incorporate painted labels and agglomerations in 3D visualization. Marching Cubes is parallelized over small blocks. Only relevant blocks are considered (huge speed-up for sparse label data).

Paintera is implemented in Java and makes extensive use of the UI framework JavaFX

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.

MTrack is a tool, which detects, tracks, and measures the behavior of fluorescently labeled microtubules imaged by TIRF (total internal reflection fluorescence) microscopy. In such an in vitro reconstitution approach, stabilized, non-dynamic microtubule seeds serve as nucleation points for dynamically growing microtubules.

MTrack is a bi-modular tool. The first module detects and tracks the growing microtubule ends and creates trajectories. The second module uses these trajectories to fit models of dynamic behavior (polymerization and depolymerization velocities, catastrophe and rescue frequencies). It also computes statistics such as length and lifetime distributions when analyzing more than one movie (batch mode).

"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)

This project was designed for vectorize and analyze the  blood vessels in the mouse brain.

This plugin requires the definition of seed point detection settings by the user (Semi-automated).

We have developed a novel approach, named DF-Tracing, to tackle this challenge. This method first extracts the neurite signal (foreground) from a noisy image by using anisotropic filtering and automated thresholding. Then, DF-Tracing executes a coupled distance-field (DF) algorithm on the extracted foreground neurite signal and reconstructs the neuron morphology automatically. Two distance-transform based “force” fields are used: one for “pressure”, which is the distance transform field of foreground pixels (voxels) to the background, and another for “thrust”, which is the distance transform field of the foreground pixels to an automatically determined seed point. The coupling of these two force fields can“push” a “rolling ball” quickly along the skeleton of a neuron, reconstructing the 3D cell morphology.

nctuTW is a "high-throughput computer method of reconstructing the neuronal structure of the fruit fly brain. The design philosophy of the proposed method differs from those of previous methods. We propose first to compute the 2D skeletons of a neuron in each slice of the image stack. The 3D neuronal structure is then constructed from the 2D skeletons. Biologists tend to use confocal microscopes for optimal images in a slice for human visualization; and images in two consecutive slices contain overlapped information. Consequently, a spherical object becomes oval in the image stack; that is, neurons in the image stack do not reflect the true shape of the neuron. This is the main reason we chose not to work directly on the 3D volume.

The proposed method comprises two steps. The first is the image processing step, which involves computing a set of voxels that is a superset of the 3D centerlines of the neuron. The shortest path graph algorithm then computes the centerlines. The proposed method was applied to process more than 16 000 neurons. By using a large amount of reconstructions, this study also demonstrated a result derived from the reconstructed data using the clustering technique." (Extracted from reference publication)

Illustrative image shows gold standard (top) and method results (bottom). 

By combining multiple image alignment and tracing into one program, Reconstruct (TM) allows images to be processed more efficiently. Tracing can be done directly on the transformed images and alignments can be asily modified. Reconstruct (TM) was developed from years of experience working with high magnification serial section images of brain tissue. (Extracted from User Manual)

"The original platform of the Reconstruct program allows a user to trace objects in serial sections by manually drawing the outline of each object on each section, which is time-consuming. We modified Reconstruct to enable semi-automatic tracing of axons using a region-growing algorithm called wildfire."

JFilament is an ImageJ plugin for segmentation and tracking of 2D and 3D filaments in fluorescenece microscopy images. The main algorithm used in Jfilament is "Stretching Open Active Contours" (SOAC). In order to use this method, the user must define seed points in the image where the SOAC method will begin.

JFilament also includes 2D "closed" active contours which can be used for tasks such as segmentation and tracking of cell boundaries.

The ultimate goal of the NET framework is to make images of networks processable by computers. Therefore we want to have a pixel based image as input, as output we want a representation of the network visible in the image that retains as much information about the original network as possible. NET achives this by first segmenting the image and then vectorizing the network and then extracting information. The information we extract is

• First and foremost the graph of the network. We find the crossings (nodes) and connections between crossings (edges) and therefore extract information about the neighborhood relations, the topology of the network.
• We also extract the coordinates of all nodes which enables us to embed them into space. We therefore extract information about the geometry of the network.
• Last but not least we track the radii of the edges in the extraction process. Therefore every edge has a radius which can be identified with its conductivity.

In the following we will first provide detailed instructions on how to install NET on several platforms. Then we describe the functionality and options of each of the four scripts that make up the NET framework.

ORION: Online Reconstruction and functional Imaging Of Neurons: segmentation and tracing of neurons for reconstruction.

A project to develop tools that explore single neuron function via sophisticated image analysis. ORION software bridges advanced optical imaging and compartmental modeling of neuronal function by rapidly, accurately, and robustly generating, from structural image data, a cylindrical morphology model suitable for simulating neuronal function. The goal of this project is to develop a computational and experimental framework to allow real-time mapping of functional imaging data (e.g., spatio-temporal patterns of dendritic voltages or intracellularions) to neuronal structure, during the very limited duration of an acute experiment.

The invention comprises a software tool, NeuronMetrics, which functions as a set of modules that run in the open-source program ImageJ. NeuronMetrics features a novel method for estimating neural “branch number” (a measure of the axonal complexity) from two-dimensional images. In addition, the tool features a novel method for modeling neural structure in large “gaps” that result from image artifacts.

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." 

AnaMorf is a plug-in developed for the ImageJ platform (rsb.info.nih.gov/ij) to analyse the microscopic morphology of filamentous microbes. The program returns average data on a population of mycelial elements, using the descriptors projected area, circularity, total hyphal length, number of hyphal tips, hyphal growth unit, lacunarity and fractal dimension. The plug-in accepts as input a user-specified directory of images, analysing each and outputing tabulated results.

This plugin tags all pixel/voxels in a skeleton image and then counts all its junctions, triple and quadruple points and branches, and measures their average and maximum length.

Tags are shown in a new window displaying every tag in a different color. You can find it under [Plugins>Skeleton>Analyze Skeleton (2D/3D)]. See Skeletonize3D for an example of how to produce skeleton images.

The voxels are classified into three different categories depending on their 26 neighbors: - End-point voxels: if they have less than 2 neighbors. - Junction voxels: if they have more than 2 neighbors. - Slab voxels: if they have exactly 2 neighbors.

End-point voxels are displayed in blue, slab voxels in orange and junction voxels in purple.

Notice here that, following this notation, the number of junction voxels can be different from the number of actual junctions since some junction voxels can be neighbors of each other.

Output data type: table result, image of the skeleton

hIPNAT (hIPNAT: Image Processing for NeuroAnatomy and Tree-like structures) is a set of tools for the analysis of images of neurons and other tree-like morphologies. It is written for ImageJ, the de facto standard in scientific image processing. It is available through the ImageJ Neuroanatomy update site.

"we propose a novel automatic 3D neuron reconstruction algorithm, named Rivulet, which is based on the multi-stencils fast-marching and iterative back-tracking. The proposed Rivulet algorithm is capable of tracing discontinuous areas without being interrupted by densely distributed noises." 

This plugin can be used with default parameters or with user-defined parameters.

Example image obtained from Rivulet Wiki website (https://github.com/RivuletStudio/Rivulet-Neuron-Tracing-Toolbox/wiki) 

"we present a new fully automated 3D reconstruction algorithm, called TReMAP, short for Tracing, Reverse Mapping and Assembling of 2D Projections. Instead of tracing a 3D image directly in the 3D space as seen in majority of the tracing methods, we first trace the 2D projection trees in 2Dplanes, followed by reverse-mapping the resulting 2D tracing results back into the 3D space as 3D curves; then we use a minimal spanning tree (MST) method to assemble all the 3D curves to generate the final 3D reconstruction. Because we simplify a 3D reconstruction problem into 2D, the computational costs are reduced dramatically." 

Suitable for high throughput neuron image analysis (image sizes >10GB). This plugin can be used with default parameters or user-defined parameters.

All-path-pruning 2.0 (APP2) is a component of Vaa3D. 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. APP2 computes the distance transform of all image voxels directly for a gray-scale image, without the need to binarize the image before invoking the conventional distance transform. APP2 uses a fast-marching algorithm-based method to compute the initial reconstruction trees without pre-computing a large graph. This method allows to trace large images. This method can be used with default parameters or user-defined parameters.

"We have developed an automatic graph algorithm, called the all-path pruning (APP), to trace the 3D structure of a neuron. To avoid potential mis-tracing of some parts of a neuron, an APP first produces an initial over-reconstruction, by tracing the optimal geodesic shortest path from the seed location to every possible destination voxel/pixel location in the image. Since the initial reconstruction contains all the possible paths and thus could contain redundant structural components (SC), we simplify the entire reconstruction without compromising its connectedness by pruning the redundant structural elements, using a new maximal- covering minimal-redundant (MCMR) subgraph algorithm. We show that MCMR has a linear computational complexity and will converge. We examined the performance of our method using challenging 3D neuronal image datasets of model organisms (e.g. fruit fly)"

This plugin can be used with default parameters or user-defined parameters.

The Sprout Morphology plugin measures sprout number, length, width and cell density of endothelial cell (EC) sprouts grown in a bead sprouting assay. It optionally includes measuring the coverage of these sprouts with pericytes included in the assay, as well as the endothelial cell/pericyte ratio.

SOAX is an open source software tool to extract the centerlines, junctions and filament lengths of biopolymer networks in 2D and 3D images. It facilitates quantitative, reproducible and objective analysis of the image data. The underlying method of SOAX uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then stretch along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments.

SOAX provides 3D visualization for exploring image data and visually checking results against the image. Quantitative analysis functions based on extracted networks are also implemented in SOAX, including spatial distribution, orientation, and curvature of filamentous structures. SOAX also provides interactive manual editing to further improve the extraction results, which can be saved in a file for archiving or further analysis. Useful for microtubules or actin filaments.

Observation: Depending on the operating system, the installation may or may not require Boost C++, ITK and VTK libraries. Windows has a standalone executable application without the need of those. 

Image-processing algorithms developed at the MOSAIC Group for fluorescence microscopy. Tools included:

• 2D/3D single-particle tracking tool which can be used to track bright spots in 2D/3D movies over time.
• Optimal filament segmentation of 2D images. 
• Curvature filters for image filtering, denoising, and restoration. 
• Image naturalization for image enhancement based on gradient statistics of natural-scence images. 
• Tool for automatically send and distribute jobs on clusters and get back the results.
• Multi-region image segmentation of 2D and 3D images without needing to know the number of regions beforehand. 
• Squassh for globally optimal segmentation of piecewise constant regions in 2D and 3D images and for object-based co-localization analysis. 
• Tool for inferring spatial interactions between patterns of objects in images or between coordinates read from a file.
• Tool for robust, histogram-based background subtraction well suited to correct for inhomogeneous illumination artifacts.
• A tool to estimate the Point-Spread Function of the microscopy out of 2D fluorescence images.
• A tool to measure the 3D Point-Spread Function of a confocal microscope from an image stack.
• Addition of synthetic Poisson-distributed noise to an image in order to simulate shot noise of various signal-to-noise ratios. 
• Convolution of an image with a Bessel function in order to simulate imaging with a microscope. 
• A utility to detect bright spots in images and estimate their center. 
• A utility to create manual segmentations to be used as ground truth to test and benchmark automatic segmentation algorithms.
• A tool for replacing one color in an image with another color.

neuTube is a collection of neuron reconstruction tools from fluorescence microscope images. It has an interactive system with a 3D viewer, which can be clicked in 3D and perform neuron tracing automatically and semi-automatically. It can automatically recognize branching points as junctions. Traced neurons can be exported to swc format, which could be imported by various software packages. neuTube has Win and Mac OS standalone executable builds and may also be installed by manual compilation. In addition, neuTube can be used as a plugin in Vaa3D.

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.

Task

Quantify the length of microtubules (MT) and the MT average density per cell.

Workflow descriptions

Simple two step workflow, allowing visual & manual correction of microtubule between the 2 steps. Batch measurement of microtubule lengths for multiple images is achieved by segmenting the MTs and then their skeletonizations. The number of pixels in the microtubule is proportional to their length, so the length can be estimated.

Script

Workflow is written as an ImageJ macro (Fiji) with following steps:

1. The enhancement of tubular structure by computing eigenvalues of the hessian matrix on a Gaussian filtered version of the image ( sigma 1 pixel), as implemented in the tubeness plugin.

2. The tubules were then thresholded , and structures containing less than 3 pixels were discarded.

3. If needed, a visual check and correction of segmented microtubule is then performed.

4. After correction, segmented MTs were then reduced to a 1-pixel thick line using the skeletonize plugin of Fiji. The length of the skeletonized microtubules was then directly proportional to their length.

5. Data were grouped by condition and converted back to micrometers units under Matlab for the statistical tests.

Pitfalls

Commented but not that general without editing some fields in the macros.

Sample Data

Sample data and workflow (see above URL) can be accessed by - login: biii - password Biii!

Misc

3D version also available here. Use of components Skeletonize and Tubeness Filter

Neuron studio is a software package to reconstruct neurons from 3D confocal images. Reconstruction can be done manually, semi-manually or fully automatic. The images as well as the detected objects are rendered in 3D. A spine detection and classification function is also included. Results can be exported as a text file with coords of the spines. It seems that active development has stopped in 2009. NeuronStudio is being developed at the Computational Neurobiology and Imaging Center (CNIC), a research laboratory at the Neuroscience Department of the Mount Sinai School of Medicine in New York.

NeuronStudio can be used with default parameters or user-defined parameters (Fully or semi-automated).

The tool measures the total length of the microtubules in a 3D image.

See: http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Microtubules_Tool_(3…

You can find a test image here.

Plugin designed to allow easy semi-automatic tracing of neurons or other filament-like structures (e.g., microtubules, blood vessels) through either 2D images or 3D image stacks. 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.

This plugin is included in Fiji by default.

The Sholl technique is used to describe neuronal arbors. This plugin can perform Sholl directly on 2D and 3D grayscale images of isolated neurons. Its internal algorithm to collect data is based upon how Sholl analysis is done by hand — it creates a series of concentric shells (circles or spheres) around the focus of a neuronal arbor, and counts how many times connected voxels defining the arbor intersect the sampling shells. The major advantages of this plugin over other implementations are:

• When analyzing images directly, it does not require previous tracing of the arbor (although it can also analyze traced arbors)
• It combines curve fitting with several methods to automatically retrieve quantitative descriptors from sampled data, which allows direct statistical comparisons between arbors
• It allows continuous and repeated sampling around user-defined foci
• It allows batch processing