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False DNA tetraploid or aneuploid peaks may be detected by FCM-DNA because of clumping of nuclei, whereas ICM-DNA allows excluding nuclei clumps during the visual inspection step of nuclei galleries (for examples, see Figure 2).
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(ii) An initial segmentation delineates isolated nuclei or clumps of nuclei from the background.
To increase computational throughput, individual nucleus and clumps of nuclei are delineated from the background using an initial segmentation.
The clustered points of curvature maxima define planes that can partition a clump of nuclei into individual nucleus.
The fluorescence from Hoechst 33342 (405 nM excitation, 425 475 nm emission) was used to select for a 2N DNA content, thus avoiding clumps of nuclei.
The output of this step is a set of plane models that will be used to partition the clumps of nuclei.
For example, our analysis reveals that the computation of the image curvature, described in section 3.4.1, may miss a clump of nuclei because of the scale of the fold between two adjacent nuclei.
The strategy uses a cascade of geometric filters that are insensitive to spatial non-uniformity and partitions a clump of nuclei based on the grouping of points of maximum curvature at the interface of two neighboring nuclei.
Meanwhile, some of the nuclei appeared small and shrunken with peripheral clumping of heterochromatin, while other nuclei showed dilated perinuclear cisternae.
As a result, the segmentation strategy initially detects isolated nuclei and groups of nuclei that are clumped together prior to subsequent partitioning.
The confocal images also show that upon peptide treatment, the cell nucleus undergoes the characteristic morphological changes associated with apoptosis, such as clumping of nuclear chromatin and condensation of nuclei.
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