A common approach is the use of a fluorescent marker to segment objects of interest (e.g. Different strategies have been used to construct 3D ROIs. Analysis in 3D becomes important when objects of interest stretch across more than one optical section. Regions of interest (ROIs) can be delineated either in two dimensions (X/Y, 2D) or three dimensions (X/Y/Z, 3D). This increases statistical power and provides fine-grained information on cell-to-cell variability. Automation minimizes subjectivity in the analysis, saves time, and allows for the quantification of more cells than manual analysis. This task is greatly aided by automated or semi-automated image analysis pipelines 1, 2, 3. However, collecting quantitative data from microscopy images represents a persistent bottleneck. Microscopy is a powerful technique to collect spatial and temporal information on cellular processes. 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 radially symmetric cell types. The pipeline is available as a macro for the open-source image analysis software Fiji/ImageJ. 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. Thus, Pomegranate accurately represents radially symmetric cells in 3D even if cell diameter varies and regardless of whether a cell is straight, bent or curved. The diameter of these spheres adapts to the cell diameter at each position. Here, we report Pomegranate, a pipeline that performs the extrusion into 3D using spheres placed along the topological skeletons of the 2D-segmented regions. However, current methods typically make the simplifying assumption that cells are straight rods. In radially symmetric cells, such as fission yeast and many bacteria, this 2D segmentation can be computationally extruded into the third dimension. However, brightfield images only readily provide information for two-dimensional (2D) segmentation. Using brightfield images for segmentation has the advantage of being minimally phototoxic and leaving all other channels available for signals of interest. Three-dimensional (3D) segmentation of cells in microscopy images is crucial to accurately capture signals that extend across optical sections.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |