Object detection

WASH, Exo84, and cortactin spot detection and codistribution analysis To detect endosomes, an automatic Otsu threshold is applied to the Gaussian-filtered MT1-MMP–positive endosome image (= 1.5 pixels for the sample image). Statistics about each endosome are then saved, for example random positioning of spots can be compared to actual positioning. For each endosome, WASH and Exo84 (or WASH and cortactin) spots are searched for in a neighboring of x pixels in their respective channel. Their number and position are saved per endosome (**see the macro in Text file S2 downloadable from here**).

From the position of WASH and Exo84 (or WASH and cortactin) spots around each endosomes, each WASH spot is paired with its closest Exo84 (or cortactin) spot neighbor, optimized over all spots around this endosome.

This allowed measuring of the distribution of distance between WASH-Exo84 (or WASH-cortactin) spots (**for the co-distribution analysis, see matlab scripts in Zip file S3 downloadable).

## Features >The IJBlob library indentifying connected components in binary images. The algorithm used for connected component labeling is: >Chang, F. (2004). A linear-time component-labeling algorithm using contour tracing technique. Computer Vision and Image Understanding, 93(2), 206–220. doi:10.1016/j.cviu.2003.09.002 ##Reference Wagner, T and Lipinski, H 2013. IJBlob: An ImageJ Library for Connected Component Analysis and Shape Analysis. Journal of Open Research Software 1(1):e6, DOI:

Imaris is a software for data visualization, analysis, segmentation and interpretation of 3D and 4D microscopy images. It performs interactive volume rendering that lets users freely navigate even very large datasets (hundreds of GB). It performs both manual and automated detection and tracking of biological “objects” such as cells, nuclei, vesicles, neurons, and many more. ImarisSpots for example is a tool to detect “spherical objects” and track them in time series. Besides the automated detection it gives the user the ability to manually delete and place new spots in 3D space. ImarisCell is a tool to detect nuclei, cell boundaries and vesicles and track these through time. ImarisFilament is a module that lets users trace neurons and detect spines. For any detected object Imaris computes a large set of statistics values such as volume, surface area, maximum intensity of first channel, number of vesicles per cell etc. These values can be exported to Excel and statistics software packages. The measurements can also be analyzed directly within ImarisVantage which is a statistics tool that provides the link back to the 3D objects and the original image data. Strengths: - good visualization - user friendly interface - reads most microscopy file formats - image analysis workflows are very easy to apply - interactive editing of objects to correct errors during automatic detection - large data visualization (hundreds of GB)

Semi-automated segmentation of 3D objects (e.g. neurons) based on user-provided seeds.