This study tests the use of a constrained nonnegative matrix factorization (NMF) algorithm to explore the comparatively new field of chemometric microscopy to support tissue diagnosis. The algorithm can extract the spectral signature and the absolute concentration map of endogenous fluorophores from wide-field microscopic images. The resultant data distinguished normal and fibrous calvarial tissues, based on the changes in their spectral signatures. The absolute concentration map of endogenous fluorophores, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and lipofuscin were derived from microscopic images and compared with the fluorescence from pure fluorophores. While the absolute concentration of NADH increased, the same of FAD and lipofuscin decreased from a normal to fibrous calvarial condition. An increase in the optical redox ratio, possibly due to the metabolic changes during the development of fibrosis, was observed. Differentiating tissue types using the absolute concentration map was found to be considerably more precise than that achievable with relative concentration. The quantification of fluorophores with reference to the absolute concentration map can eliminate uncertainties due to system responses or measurement details, thereby generating more biologically apposite data. Wide-field microscopy augmented with a constrained NMF algorithm could emerge as an advanced diagnostic tool, potentially heralding the emergence of chemometric microscopy.

Weedy red rice is a highly troublesome weed of rice in the United States and throughout the world. Effective management of this weed has remained challenging to U.S. farmers, partly because of the biological diversity among red rice populations, resistance to or avoidance of control measures, and genetic similarities with crop rice that allow crossing between the two plant types. The aim of this research was to identify simple sequence repeat (SSR) marker loci that will unambiguously differentiate between U.S. weedy red rice, commercial rice cultivars, and their hybrids, to characterize the genetic diversity and structure of U.S. weedy red rice accessions in relation to Oryza collections from international sources, and to relate genetic and geographic variability within U.S. weedy red rice. Thirty-one SSR markers were used to analyze 180 worldwide Oryza entries and 80 U.S. weedy red rice and U.S. rice cultivars. Twenty-six of the 31 SSR marker loci were highly informative with respect to genetic distinctions between U.S. weedy red rice and U.S. rice cultivars. U.S. red rice are accessions clustered into two main SSR-based collections, awnless strawhull (SA−) and awned blackhull (BA+), according to genetic distance analysis and principal coordinate analysis. Genetic structure analysis clearly identified SA− and BA+ red rice, rice–red rice hybrids, commercial japonica rice cultivars, indica rice, and a number of international and wild Oryza spp. standards (e.g., Oryza nivara, Oryza rufipogon, and Oryza glaberrima) as genetically distinct groups. U.S. SA− red rice exhibited greater spatial structure than did BA+ in that the genetic makeup of SA− accessions changed nearly twice as much with geographic distance as compared to BA+. However, the overall genetic variability within SA− red rice accessions was less than for BA+ accessions, suggesting that the SA− types may be genetically less compatible than BA+ types with other Oryza plants such as rice or other red rice types present in U.S. rice fields. Several of the awned red rice entries exhibited evidence of natural hybridization with different red rice types. Our results suggest that the SA− and BA+ red rice collections have different genetic backgrounds. SA− accessions generally associated most closely with indica-like red- or white-bran Oryza sativa cultivar standards, while BA+ accessions generally associated more closely with O. nivara or O. nivara–like O. sativa entries. Although the U.S. red rice accessions appear not to have descended directly from introductions of the worldwide Oryza standards analyzed, an Oryza red-pericarp entry from Niger (UA 1012; PI 490783) was genetically very similar to some U.S. BA+ accessions.