Object-based colocalisation

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Measuring the colocalization between fluorescently labeled molecules is a widely used approach to measure the degree of spatial coincidence and potential interactions among subcellular species (e.g., proteins). This example shows how the object identification and RelateObjects modules are used to measure the degree of overlap between two fluorescent channels. Sample image is included in the download package.

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).

The Huygens Software Suite consists of different image processing packages with functionalities that include deconvolution, interactive analysis, and volume visualization of 2D-3D multi-channel and time series images from fluorescence microscopes such as widefield, confocal, multi-photon, spinning disk, Array Detector, STED, and Light Sheet

‘’’Squassh’’’ is a tool for 2D and 3D segmentation and quantification of subcellular shapes in fluorescence microscopy images. It provides globally optimal detection and segmentation of objects with constant internal intensity distribution, followed by object-based colocalization analysis. The segmentation computed by Region Competition can optionally correct for the PSF of the microscope, hence providing optimally deconvolved segmentations. Part of the mosaic suite