Type I collagen is a major component of the extracellular matrix as well as many tissue engineered models. To understand changes in collagen related models over time, it is important to evaluate collagen dynamics with noninvasive techniques. Fluorescence spectroscopy provides a method to noninvasively measure endogenous collagen fluorescence. Additionally, second harmonic generation (SHG) imaging of collagen produces high resolution images of the fibrils. In this study, a novel in vitro collagen measurement chamber was developed for measurement in standard spectroscopic cuvette chambers and microscopic imaging. The fluorescence of polymerized collagen was found to be highly variable, primarily depending on incubation time after polymerization. Changes in fluorescence over time were consistent with increases at UVA excitation wavelengths (ex=360 nm) and decreases at UVC excitation wavelengths (ex=270 nm), suggesting changes in nonenzymatic association of the collagen fibrils. SHG imaging of the collagen suggested that a stable network formed during polymerization. Unlike the fluorescence emission, SHG images from the gels varied little with time suggesting that SHG is not as sensitive to cross-linking or fibril-fibril associated changes. The developed measurement system will allow further studies on the effect of enzymatic cleavages and structural alterations on collagen fluorescence and SHG.
In order to understand the distribution of endogenous fluorescence in the ovary, ovarian biopsies were maintained with a viable tissue imaging system and characterized with multiphoton imaging. It was imperative to maintain a stable in vitro environment so that tissue images could provide accurate correlative data for in vivo spectroscopic measurements. Evaluating tissue viability in real time poses a difficult task given that viability assays are tailored for cell culture. The focus of this study was to design a robust in vitro imaging chamber for assessment of ovarian autofluorescence and simple, reliable viability assays for tissue status monitoring.
Non-invasive monitoring of cellular metabolism offers promising insights into areas ranging from biomarkers for drug activity to cancer diagnosis. Fluorescence spectroscopy can be utilized in order to exploit endogenous fluorophores, typically metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and estimate the redox status of the sample. Fluorescence spectroscopy was applied to follow metabolic changes in epithelial ovarian cells as well as bladder epithelial cancer cells during treatment with a chemopreventive drug that initiates cellular quiescence. Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity, redox status, and protein content. Cells were treated with varying concentrations of N-4-(hydroxyphenyl) retinamide (4-HPR) and measured in a stable environment with a sensitive fluorescence spectrometer. A subset of measurements was completed on a low concentration of cells to demonstrate feasibility for medical application such as in bladder or ovary washes. Results suggest that all of the cells responded with similar dose dependence but started at different estimated redox ratio baseline levels correlating with cell cycle, growth inhibition, and apoptosis assays. NADH and tryptophan related fluorescence changed significantly while FAD related fluorescence remained unaltered. Fluorescence data collected from approximately 1000 - 2000 cells, comparable to a bladder or ovary wash, was measurable and useful for future experiments. This study suggests that future intrinsic biomarker measurements may need to be most sensitive to changes in NADH and tryptophan related fluorescence while using FAD related fluorescence to help estimate the baseline redox ratio and predict response to chemopreventive agents.
Epithelial ovarian cancer has the highest mortality rate among the gynecologic cancers. Early detection would significantly improve survival and quality of life of women at increased risk to develop ovarian cancer. We have constructed a device to investigate endogenous signals of the ovarian tissue surface in the UV C to visible range and describe our initial investigation of the use of optical spectroscopy to characterize the condition of the ovary. We have acquired data from more than 33 patients. A table top spectroscopy system was used to collect endogenous fluorescence with a fiberoptic probe that is compatible with endoscopic techniques. Samples were broken into five groups: Normal-Low Risk (for developing ovarian cancer) Normal-High Risk, Benign, and Cancer. Rigorous statistical analysis was applied to the data using variance tests for direct intensity versus diagnostic group comparisons and principal component analysis (PCA) to study the variance of the whole data set. We conclude that the diagnostically most useful excitation wavelengths are located in the UV. Furthermore, our results indicate that UV B and C are most useful. A safety analysis indicates that UV-C imaging can be conducted at exposure levels below safety thresholds. We found that fluorescence excited in the UV-C and UV-B range increases from benign to normal to cancerous tissues. This is in contrast to the emission created with UV-A excitation which decreased in the same order. We hypothesize that an increase of protein production and a decrease of fluorescence contributions of the extracellular matrix could explain this behavior. Variance analysis also identified fluctuation of fluorescence at 320/380 which is associated with collagen cross link residues. Small differences were observed between the group at high risk and normal risk for ovarian cancer. High risk samples deviated towards the cancer group and low risk samples towards benign group.