Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment.

Today's advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study, topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n = 163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler's (K-L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar, and dissimilar. Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (approximately 100%), 5-AZA-treated cells (90%), and a lower degree of same type of cells (64%) in the OCT-treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e., the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K-L divergence has proven to be a more valuable and robust tool for categorization of individual cells within a population, with potential applications in epigenetic drug screening.

Polarization-sensitive hyperspectral imaging in vivo: a multimode dermoscope for skin analysis.

Attempts to understand the changes in the structure and physiology of human skin abnormalities by non-invasive optical imaging are aided by spectroscopic methods that quantify, at the molecular level, variations in tissue oxygenation and melanin distribution. However, current commercial and research systems to map hemoglobin and melanin do not correlate well with pathology for pigmented lesions or darker skin. We developed a multimode dermoscope that combines polarization and hyperspectral imaging with an efficient analytical model to map the distribution of specific skin bio-molecules. This corrects for the melanin-hemoglobin misestimation common to other systems, without resorting to complex and computationally intensive tissue optical models. For this system's proof of concept, human skin measurements on melanocytic nevus, vitiligo, and venous occlusion conditions were performed in volunteers. The resulting molecular distribution maps matched physiological and anatomical expectations, confirming a technologic approach that can be applied to next generation dermoscopes and having biological plausibility that is likely to appeal to dermatologists.

Spectral imaging for precise surgical intervention in Hirschsprung's disease.

We used advanced spectral imaging for intrasurgical decision making in a preclinical study, on a mouse model of Hirschsprung's Disease. Our imaging device sampled areas from normal and abnormal (aganglionic) colon in these animals. Spectral segmentation and classification of the resulting images showed a clear distinction between the normal and aganglionic regions, as confirmed by pathological analysis and use of mutant mice. We developed a simple algorithm that could distinguish normal from aganglionic colon with high spatial resolution and reproducibility, and the following statistics: sensitivity = 97%, specificity = 94%, positive predictive value = 92%, negative predictive value = 98%. These studies showed translational proof of concept that spectral imaging could be used during operations, in real time, to help surgeons precisely distinguish normal from abnormal tissue without requiring traditional biopsy.

In vivo cytometry: a spectrum of possibilities.

BACKGROUND: We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex-vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real-time, high resolution intrasurgical optical diagnostics. METHODS: We use reflectance, fluorescence, two-photon, and Mie scattering imaging, performed with instrumentation we developed or modified, to detect intrinsic tissue signatures. Emphasis is on spectral/hyperspectral imaging approaches allowing the equivalent of in vivo pathology. RESULTS: With experimental focus on unstained specimens, we demonstrate the ability to segment tissue images for cancer detection. Spectral reflectance imaging, coupled with advanced analysis, typically yields 90% specificity and sensitivity. Autofluorescence is also shown to be diagnostically useful, with lymph nodes results highlighted here. Elastic scattering hyperspectral imaging endoscopy, using a new instrument we designed and built, shows promise in bronchoscopic detection of dysplasia and early cancer in patients. CONCLUSIONS: The results demonstrate that advanced optical imaging can detect and localize cellular signatures of cancer in real-time, in vivo, without the use of contrast agents, in animals and humans. This is an important step towards tight spatio-temporal coupling between such detection and clinical intervention.