Graphical methods for quantifying macromolecules through bright field imaging.

Abstract

Bright field imaging of biological samples stained with antibodies and/or special stains provides a rapid protocol for visualizing various macromolecules. However, this method of sample staining and imaging is rarely employed for direct quantitative analysis due to variations in sample fixations, ambiguities introduced by color composition and the limited dynamic range of imaging instruments. We demonstrate that, through the decomposition of color signals, staining can be scored on a cell-by-cell basis. We have applied our method to fibroblasts grown from histologically normal breast tissue biopsies obtained from two distinct populations. Initially, nuclear regions are segmented through conversion of color images into gray scale, and detection of dark elliptic features. Subsequently, the strength of staining is quantified by a color decomposition model that is optimized by a graph cut algorithm. In rare cases where nuclear signal is significantly altered as a result of sample preparation, nuclear segmentation can be validated and corrected. Finally, segmented stained patterns are associated with each nuclear region following region-based tessellation. Compared to classical non-negative matrix factorization, proposed method: (i) improves color decomposition, (ii) has a better noise immunity, (iii) is more invariant to initial conditions and (iv) has a superior computing performance.