2D

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wider range of blinking conditions. Balanced SOFI analyses several cumulant orders for extracting molecular parameter maps, such as the bright and dark state lifetimes, the concentration and the brightness distributions of fluorophores within biological samples. In combination with a deconvolution of the cumulant images, the estimated parameter maps proved useful to balance the image contrast and to linearize the brightness and blinking response. Thereby, the image quality and the resolution were improved significantly.

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.

arivis Vision4D is a modular software for working with multi-channel 2D, 3D and 4D images of almost unlimited size independent of available RAM. Many imaging systems, such as high speed confocal, Light Sheet/ SPIM and 2 Photon systems, can produce a huge amount of multi-channel data, which arivis Vision4D handles without constraints. Terabyte ready arivis Vision4D main functionality: Easy import of most image formats from microsopes as well as biological formats High performance interactive 3D / 4D rendering on standard PCs and laptops with 3D Graphics Support Intuitive tools for stitching and alignment to create large multi-dimensional image stacks Immediate 2D, 3D and 4D visualization, annotation and analysis regardless of image size Creation, import, and export of 4D Iso-surfaces Powerful Analysis Pipeline for 3D /4D image analysis (cell segmentation, tracking, annotation, quantitative measurement and statistics, etc) Semi-automatic/manual segmentation and tracking with interactive Track Editor Easy design and export of 3D / 4D High Resolution Movies Seamless integration of custom workflows via Matlab API and Python scripting Data sharing for collaboration A user friendly software, easy to learn and use for any life scientist

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

ThunderSTORM is an open-source, interactive, and modular plug-in for ImageJ designed for automated processing, analysis, and visualization of data acquired by single molecule localization microscopy methods such as PALM and STORM. Our philosophy in developing ThunderSTORM has been to offer an extensive collection of processing and post-processing methods so that users can easily adapt the process of analysis to their data.