Automated 3D bio-imaging analysis of nuclear organization by NucleusJ 2.0

Abstract

NucleusJ 1.0, an ImageJ plugin, has been shown to be a useful tool to analyse nuclear morphology and chromatin organisation in plant and animal cells. However, technological improvements of confocal microscopy have speeded-up image acquisition, highlighting the bottleneck in 3D image analysis caused by manual steps in NucleusJ 1.0 and limiting its use for big data analysis. NucleusJ 2.0 is a new release of NucleusJ, in which image processing is achieved more quickly using a command-line user interface. Starting with large collection of 3D nuclei, segmentation can be performed by the previously developed Otsu-modified method or by a new 3D gift-wrapping method, taking better account of nuclear indentations and unstained nucleoli. These two complementary methods based on threshold and edgebased detection, are compared for their accuracy by using three types of datasets-digitised spheres, microspheres and plant nuclei-available to the community through an open source repository accessible at https://www.brookes.ac.uk/indepth/images/. A discrete geometric method was introduced to improve the surface area calculation, a key parameter when studying nuclear morphology, replacing an ImageJ default tool by a new one that includes pixel context information. Finally, NucleusJ 2.0 was evaluated using original plant genetic material, in which nuclear morphology and chromatin organisation are strongly affected and by assessing its efficiency on nuclei stained with DNA dyes or after 3D-DNA Fluorescence in situ hybridisation. With these improvements, NucleusJ 2.0 permits the generation of large user-curated datasets that will be useful for software benchmarking or to train convolution neural networks.

Methods forin situquantitative root biology

“…We found rapid divergence of the nuclei volume and nuclei heterogeneity between cell types even in the division zone of the RAM, with higher nuclei volume in the cortex and epidermis layers (Figure 2C). Next, we extended our analysis to single nuclei features by using the NucleusJ2.0 plugin that allowed us to extract up to 12 morphological parameters per nucleus (Dubos et al 2020). When studying cells of the same tissue layer in the PR, we can distinguish different cell cycle stages in the proliferation zone of the RAM based on nuclei volume and other morphological features (Figure 2D, E).…”

“…Thereafter nucleus edges have been detected, images were converted to hdf5 and further segmentation was performed accordingly to the standard pipeline (Pasternak et al 2021b). Individual nuclei analyses were performed according to Dubos et al (2020) with slight modifications. Single layer images from a given cell type and area containing 20-30 nuclei have been selected, and processed with the NucleusJ2.0 plugin as described elsewhere (Dubos et al 2020).…”

“…Individual nuclei analyses were performed according to Dubos et al (2020) with slight modifications. Single layer images from a given cell type and area containing 20-30 nuclei have been selected, and processed with the NucleusJ2.0 plugin as described elsewhere (Dubos et al 2020).…”

Regulation and Physiological Significance of the Nuclear Shape in Plants

“…To quantify the nuclear shape and size more precisely, it is necessary to obtain and analyze a stack of three-dimensional images. NucleusJ, an ImageJ plugin, a useful tool to analyze nuclear morphology and chromatin organization, was released ( Poulet et al, 2015 ) and updated ( Dubos et al, 2020 ). The quantification of the nuclear shape is not only convincingly showing that the nucleus of some mutants is abnormal compared to the wild-type (WT) nucleus but also used to analyze the nuclear mechanical properties ( Hampoelz and Lecuit, 2011 ).…”

Editorial for the SEB 2020 special issue ‘dynamic organisation of the nucleus across kingdoms’

“…They show for the first time that the active positioning of the vegetative nucleus in proximity to the pollen tube tip is required for response to reactive oxygen species (ROS) and for Ca 2+ signaling; this is interestingly directly associated with the role of KASH proteins of the WIP family in nuclear positioning. In the second paper, Dubos et al [7] build on work to develop automated systems to analyze the shape, structure and function of the nucleus through the publicly available ImageJ plug-in NucleusJ 2.0. Developed initially for application in plants, this plug-in is appropriate for the analysis of images of the nucleus across kingdoms.…”