Free but not open source

Free-D is a three-dimensional (3D) reconstruction and modeling software. It allows to generate, process and analyze 3D point and surface models from stacks of 2D images. Free-D is an integrated software, offering in a single graphical user interface all the functionalities required for 3D modeling. It runs on Linux, Windows, and MacOS. Free-D is developed by the Modeling and Digital Imaging team of the Institut Jean-Pierre Bourgin, INRA Versailles, France.

Free-D (http://free-d.versailles.inra.fr/) is a 3D reconstruction and modeling software. It is multiplatform, free (but not open source) tool for academic research and teaching.

Here is how to proceed, using Free-D:

1. Segmentation:

* load (a collection of) individual 3d stacks

* (optional for serial sections) perform a 2D registration to align image slices

* segment/reconstruct 3D contours using snakes

* segment 3D spots

2. Construct average cell:

* normalize the contours to compute a average cell, by registering/warping 3D contours/surfaces

3. Quantification:

* project each individual cell to the average one

* build density maps to analyze (cartography)

A few notes for current software version (till 10/2016):

* input file format: tiff (not able to import bioformats)

* currently results are saved in customized format, but there is an exportor to convert this format into fiji readable one

* import already generated contours is on the software's TODO list

When trying to isolate objects, one strategy might be to use regular morphological operations (opening/closing) to remove small objects that are not of interest. In case small objects are made of a large number of pixels, this operation might impair the remaining objects' contours. An alternative strategy might be to use morphological reconstruction. In short, seed is placed on the image, on objects, then conditional dilation is performed from those seeds.

Here is how to proceed, using MorphoLibJ:

1. Open an image
2. Use the multi-point selection tool and place seeds on objects of interest
3. Create a new image of same size, black background
4. Transfer the selection to the new image (Edit/Selection/Restore selection)
5. Draw (make sure you're using white foreground) the multiple point selection
6. Launch the Morphological reconstruction plugin: Plugins > MorphoLibJ > Morphological reconstruction

idTracker is a videotracking software that keeps the correct identity of each individual during the whole video. It works for many animal species including mice, insects (Drosophila, ants) and fish (zebrafish, medaka, stickleback). idTracker distinguishes animals even when humans cannot, such as for size-matched siblings, and reidentifies animals after they temporarily disappear from view or across different videos. It is robust, easy to use and general. Technique details and analyses of several applications are described in Pérez-Escudero et al (2014).

Video protocol: https://www.youtube.com/watch?v=oC9tp5TKAyw

Example image: Example video of 5 zebrafish

The website implements a set of computer vision algorithms designed to automatically process time-lapse images of fluorescently labeled focal adhesion proteins in motile cells.

The methods associated with the processing have been published in PLOS One and Cell. The publication describes a quantitative analysis of focal adhesion dynamics that have been imaged using TIRF. All image processing steps are well explained or referenced.

To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones.