Segmentation of nuclei and cells using membrane related protein markers.

Segmenting individual cell nuclei from microscope images normally involves volume labelling of the nuclei with a DNA stain. However, this method often fails when the nuclei are tightly clustered in the tissue, because there is little evidence from the images on where the borders of the nuclei are. In this paper we present a method which solves this limitation and furthermore enables segmentation of whole cells. Instead of using volume stains, we used stains that specifically label the surface of nuclei or cells: lamins for the nuclear envelope and alpha-6 or beta-1 integrins for the cellular surface. The segmentation is performed by identifying unique seeds for each nucleus/cell and expanding the boundaries of the seeds until they reach the limits of the nucleus/cell, as delimited by the lamin or integrin staining, using gradient-curvature flow techniques. We tested the algorithm using computer-generated objects to evaluate its robustness against noise and applied it to cells in culture and to tissue specimens. In all the cases that we present the algorithm gave accurate results.

The Caenorhabditis elegans dosage compensation machinery is recruited to X chromosome DNA attached to an autosome.

The dosage compensation machinery of Caenorhabditis elegans is targeted specifically to the X chromosomes of hermaphrodites (XX) to reduce gene expression by half. Many of the trans-acting factors that direct the dosage compensation machinery to X have been identified, but none of the proposed cis-acting X chromosome-recognition elements needed to recruit dosage compensation components have been found. To study X chromosome recognition, we explored whether portions of an X chromosome attached to an autosome are competent to bind the C. elegans dosage compensation complex (DCC). To do so, we devised a three-dimensional in situ approach that allowed us to compare the volume, position, and number of chromosomal and subchromosomal bodies bound by the dosage compensation machinery in wild-type XX nuclei and XX nuclei carrying an X duplication. The dosage compensation complex was found to associate with a duplication of the right 30% of X, but the complex did not spread onto adjacent autosomal sequences. This result indicates that all the information required to specify X chromosome identity resides on the duplication and that the dosage compensation machinery can localize to a site distinct from the full-length hermaphrodite X chromosome. In contrast, smaller duplications of other regions of X appeared to not support localization of the DCC. In a separate effort to identify cis-acting X recognition elements, we used a computational approach to analyze genomic DNA sequences for the presence of short motifs that were abundant and overrepresented on X relative to autosomes. Fourteen families of X-enriched motifs were discovered and mapped onto the X chromosome.

Applying watershed algorithms to the segmentation of clustered nuclei.

Cluster division is a critical issue in fluorescence microscopy-based analytical cytology when preparation protocols do not provide appropriate separation of objects. Overlooking clustered nuclei and analyzing only isolated nuclei may dramatically increase analysis time or affect the statistical validation of the results. Automatic segmentation of clustered nuclei requires the implementation of specific image segmentation tools. Most algorithms are inspired by one of the two following strategies: 1) cluster division by the detection of internuclei gradients; or 2) division by definition of domains of influence (geometrical approach). Both strategies lead to completely different implementations, and usually algorithms based on a single view strategy fail to correctly segment most clustered nuclei, or perform well just for a specific type of sample. An algorithm based on morphological watersheds has been implemented and tested on the segmentation of microscopic nuclei clusters. This algorithm provides a tool that can be used for the implementation of both gradient- and domain-based algorithms, and, more importantly, for the implementation of mixed (gradient- and shape-based) algorithms. Using this algorithm, almost 90% of the test clusters were correctly segmented in peripheral blood and bone marrow preparations. The algorithm was valid for both types of samples, using the appropriate markers and transformations.