an ImageJ tool for object-based 3D co-localization and distance analysis. Methods, Elsevier, 2016, 115, pp.55-64. 10.1016/j.ymeth.2016 1 DiAna, an ImageJ tool for object-based 3D co-localization and distance analysis AbstractWe present a new plugin for ImageJ called DiAna, for Distance Analysis, which comes with a userfriendly interface. DiAna proposes robust and accurate 3D segmentation for object extraction. The plugin performs automated object-based co-localization and distance analysis. DiAna offers an indepth analysis of co-localization between objects and retrieves 3D measurements including colocalizing volumes and surfaces of contact. It also computes the distribution of distances between objects in 3D. With DiAna, we furthermore introduce an original method, which allows for estimating the statistical significance of object co-localization. DiAna offers a complete and intuitive 3D image analysis tool for biologists.

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.