Automated Biochemical, Morphological, and Organizational Assessment of Precancerous Changes from Endogenous Two-Photon Fluorescence Images

Abstract

BackgroundMulti-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques.Methodology/Principal FindingsIn this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation.Conclusions/SignificanceA wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients.

Advances and challenges in label-free nonlinear optical imaging using two-photon excitation fluorescence and second harmonic generation for cancer research

“…Majority of the ex vivo studies that investigated the redox ratio observed a decrease in the redox ratio [76,105,106] 5. In vivo auto fluorescent emission spectra of stratum corneum in normal oral cavity as compared to moderate and severe dysplasia of the oral cancer model in hamster (excitation wavelength = 780 nm).…”

“…So it would be erroneous to generalize the ratio for the entire tumour volume, while only interpreting data from the surface of the lesion. Certain in vivo and ex vivo label-free NLOI studies have observed the redox ratio to decrease with depth [103,105] as the more metabolically active proliferative cells are located deeper. However no concrete studies have been performed on the regional or spatial variation of redox ratios within a tumour and normal tissue as such.…”

Endogenous Two-Photon Fluorescence Imaging Elucidates Metabolic Changes Related to Enhanced Glycolysis and Glutamine Consumption in Precancerous Epithelial Tissues

“…Two-photon excited fluorescence (TPEF) microscopy is well-suited for such studies because of its high resolution in 3-dimensions and the ability to noninvasively detect morphologic and functional cellular changes in living tissues (6–9). This is particularly true for metabolic studies, as two key metabolic coenzymes, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), are naturally fluorescent (10) and serve as optical biomarkers to estimate the redox state of a cell (11).…”

Characterizing diabetic wound metabolism and microstructure using multi-photon microscopy

“…Low redox ratios have previously been associated with hypoxia, increased glucose consumption, and processes with significant biosynthetic demands [2,6]. The overall lower redox ratio in diabetic skin may be related to either increased glucose catabolism and/or hypoxia, but additional work is required to determine the underlying source of this optical sensitivity.…”

“…Through these techniques, collagen, keratin, and metabolic cofactors (NADH and FAD) can be detected without exogenous dyes or stains [2,3]. The overall objective of this study was to identify potential quantitative optical biomarkers capable of detecting and characterizing differences between non-diabetic and diabetic wounds.…”