Pomegranate: 2D segmentation and 3D reconstruction for fission yeast and other radially symmetric cells

Abstract

<jats:title>Abstract</jats:title><jats:p>Three-dimensional (3D) segmentation of cells in microscopy images is crucial to accurately capture signals that extend across optical sections. Using brightfield images for segmentation has the advantage of being minimally phototoxic and leaving all other channels available for signals of interest. However, brightfield images readily only provide two-dimensional (2D) segmentation. In radially symmetric cells, such as fission yeast and many bacteria, the brightfield 2D segmentation can be computationally extruded into the third dimension. However, current methods make simplifying assumptions that introduce error. Here, we report Pomegranate, a pipeline that performs the extrusion using spheres placed along the topological skeletons of the 2D-segmented regions. The diameter of these spheres adapts to the cell diameter at each position. Thus, Pomegranate accurately represents cells in 3D even if cell diameter varies and regardless of whether a cell is straight, bent or curved. We have tested Pomegranate on fission yeast and demonstrate its ability to 3D segment wild-type cells as well as classical size and shape mutants. The pipeline is available as macro for the open-source image analysis software Fiji/ImageJ. 2D segmentations created within or outside Pomegranate can serve as input, thus making this a valuable extension to the image analysis portfolio already available for fission yeast and other rod-shaped cell types.</jats:p>

Mitotic checkpoint gene expression is tuned by coding sequences

“…To analyze if and to what extent the strong mRNA variation propagates to the protein level, we quantified GFP-tagged Mad1, Mad2 and Mad3 in single cells using our 'Pomegranate' image analysis pipeline, which allows for 3D segmentation (Fig. S2, S3A) (Baybay et al, 2020). To subtract 7 autofluorescence, we mixed the GFP-expressing cells with cells not expressing GFP (Fig.…”

“…All images were deconvolved using SoftWoRx software. To correct for uneven illumination, deconvolved fluorescence images were flatfielded individually for each channel using a custom FIJI script (Baybay et al, 2020).…”

“…The Pomegranate image analysis pipeline (Baybay et al, 2020) was used to segment nuclei (using TetR-tdTomato-NLS) and whole cells (using brightfield signal and spherical extrusion of the midplane segmentation) (Fig. S2A).…”

“…S2A). We corrected for chromatic aberration and for stretching of distances in the Z direction (Baybay et al, 2020). Further analysis was conducted in R (R-Core-Team, 2020) and figures were produced using the package ggplot2 (Wickham, 2016).…”