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.

Drosophila body pigmentation has emerged as a major Evo-Devo model. Using two Drosophila melanogaster lines, Dark and Pale, selected from a natural population, we analyse here the interaction between genetic variation and environmental factors to produce this complex trait. Indeed, pigmentation varies with genotype in natural populations and is sensitive to temperature during development. We demonstrate that the bric à brac (bab) genes, that are differentially expressed between the two lines and whose expression levels vary with temperature, participate in the pigmentation difference between the Dark and Pale lines. The two lines differ in a bab regulatory sequence, the dimorphic element (called here bDE). Both bDE alleles are temperature-sensitive, but the activity of the bDE allele from the Dark line is lower than that of the bDE allele from the Pale line. Our results suggest that this difference could partly be due to differential regulation by AbdB. bab has been previously reported to be a repressor of abdominal pigmentation. We show here that one of its targets in this process is the pigmentation gene tan (t), regulated via the tan abdominal enhancer (t_MSE). Furthermore, t expression is strongly modulated by temperature in the two lines. Thus, temperature sensitivity of t expression is at least partly a consequence of bab thermal transcriptional plasticity. We therefore propose that a gene regulatory network integrating both genetic variation and temperature sensitivity modulates female abdominal pigmentation. Interestingly, both bDE and t_MSE were previously shown to have been recurrently involved in abdominal pigmentation evolution in drosophilids. We propose that the environmental sensitivity of these enhancers has turned them into evolutionary hotspots.

Half-sandwich complexes of iridium­(III) are currently being developed as anticancer drug candidates. In this context, we introduce IrBDP for which the C^N chelating phenyloxazoline ligand carries a fluorescent and lipophilic BODIPY reporter group, designed for intracellular tracking and hydrophobic compartment tropism. High-resolution analysis of cells cultured with IrBDP showed that it quickly permeates the plasma membrane and accumulates in the mitochondria and endoplasmic reticulum (ER), generating ER stress, dispersal of the Golgi apparatus, cell proliferation arrest and apoptotic cell death. Moreover, IrBDP forms fluorescent adducts with a subset of amino acids, namely histidine and cysteine, via coordination of N or S donor atoms of their side chains. Consistently, in vivo formation of covalent adducts with specific proteins is demonstrated, providing a molecular basis for the observed cytotoxicity and cellular response. Collectively, these results provide a new entry to the development of half-sandwich iridium-based anticancer drugs.

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

Modern microscopy is based on reproducible quantitative analysis, image data should be batch-processed by a standardized system that can be shared and easily reused by others. Furthermore such system should require none or minimal programming from the users. We developed TAPAS (Towards an Automated Processing and Analysis System). The goal is to design an easy system for describing and exchanging processing workflows. The protocols are simple text files comprising a linear list of commands used to process and analyse the images. An extensive set of 60 modules is already available, mostly based on the tools proposed in the 3D ImageJ Suite. We propose a wizard, called TAPAS menu, to help the user design her protocol by listing the available modules and the parameters associated. Most modules will have default parameters values for most common tasks. Once the user has designed her protocol, she can apply the protocol to a set of images, that can be either stored locally or on a OMERO database. An extensive documentation including the list of modules, various tutorials and link to the source code is available at https://imagej.net/TAPAS.

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Despite their barrier function, epithelial layers can locally lose their integrity to create physiological openings during morphogenesis. The cellular and molecular mechanisms driving the formation of these epithelial breaks are only starting to be investigated. Here, we studied the formation of the zebrafish nostril (the olfactory orifice), which opens in the skin epithelium to expose the olfactory neurons to external odorant cues. Combining live imaging, drug treatments, laser ablation and tissue-specific functional perturbations, we demonstrate that the formation of the orifice is driven by a mechanical interplay between the olfactory placode neurons and the skin: the neurons pull on the overlying skin cells in an actomyosin-dependent manner, thus triggering the opening of the orifice. This work unravels an original mechanism to break an epithelial sheet, in which an adjacent group of cells instructs and mechanically assists the epithelium to induce its local rupture.