Cell-based quantification of molecular biomarkers in histopathology specimens.

AIMS: To investigate the use of a computer-assisted technology for objective, cell-based quantification of molecular biomarkers in specified cell types in histopathology specimens, with the aim of advancing current visual estimation and pixel-level (rather than cell-based) quantification methods. METHODS AND RESULTS: Tissue specimens were multiplex-immunostained to reveal cell structures, cell type markers, and analytes, and imaged with multispectral microscopy. The image data were processed with novel software that automatically delineates and types each cell in the field, measures morphological features, and quantifies analytes in different subcellular compartments of specified cells.The methodology was validated with the use of cell blocks composed of differentially labelled cultured cells mixed in known proportions, and evaluated on human breast carcinoma specimens for quantifying human epidermal growth factor receptor 2, estrogen receptor, progesterone receptor, Ki67, phospho-extracellular signal-related kinase, and phospho-S6. Automated cell-level analyses closely matched human assessments, but, predictably, differed from pixel-level analyses of the same images. CONCLUSIONS: Our method reveals the type, distribution, morphology and biomarker state of each cell in the field, and allows multiple biomarkers to be quantified over specified cell types, regardless of their abundance. It is ideal for studying specimens from patients in clinical trials of targeted therapeutic agents, for investigating minority stromal cell subpopulations, and for phenotypic characterization to personalize therapy and prognosis.

Fast Graph Partitioning Active Contours for Image Segmentation Using Histograms.

We present a method to improve the accuracy and speed, as well as significantly reduce the memory requirements, for the recently proposed Graph Partitioning Active Contours (GPACs) algorithm for image segmentation in the work of Sumengen and Manjunath (2006). Instead of computing an approximate but still expensive dissimilarity matrix of quadratic size, ( N s 2 M s 2 ) ∕ ( n s m s ) , for a 2D image of size Ns ×Ms and regular image tiles of size ns ×ms , we use fixed length histograms and an intensity-based symmetric-centrosymmetric extensor matrix to jointly compute terms associated with the complete NsMs × NsMs dissimilarity matrix. This computationally efficient reformulation of GPAC using a very small memory footprint offers two distinct advantages over the original implementation. It speeds up convergence of the evolving active contour and seamlessly extends performance of GPAC to multidimensional images.

Cell segmentation using coupled level sets and graph-vertex coloring.

Current level-set based approaches for segmenting a large number of objects are computationally expensive since they require a unique level set per object (the N-level set paradigm), or [log2N] level sets when using a multiphase interface tracking formulation. Incorporating energy-based coupling constraints to control the topological interactions between level sets further increases the computational cost to O(N2). We propose a new approach, with dramatic computational savings, that requires only four, or fewer, level sets for an arbitrary number of similar objects (like cells) using the Delaunay graph to capture spatial relationships. Even more significantly, the coupling constraints (energy-based and topological) are incorporated using just constant O(1) complexity. The explicit topological coupling constraint, based on predicting contour collisions between adjacent level sets, is developed to further prevent false merging or absorption of neighboring cells, and also reduce fragmentation during level set evolution. The proposed four-color level set algorithm is used to efficiently and accurately segment hundreds of individual epithelial cells within a moving monolayer sheet from time-lapse images of in vitro wound healing without any false merging of cells.

QUANTITATIVE CELL MOTILITY FOR IN VITRO WOUND HEALING USING LEVEL SET-BASED ACTIVE CONTOUR TRACKING.

Quantifying the behavior of cells individually, and in clusters as part of a population, under a range of experimental conditions, is a challenging computational task with many biological applications. We propose a versatile algorithm for segmentation and tracking of multiple motile epithelial cells during wound healing using time-lapse video. The segmentation part of the proposed method relies on a level set-based active contour algorithm that robustly handles a large number of cells. The tracking part relies on a detection-based multiple-object tracking method with delayed decision enabled by multi-hypothesis testing. The combined method is robust to complex cell behavior including division and apoptosis, and to imaging artifacts such as illumination changes.