Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia¶

Abstract

Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 +/- 39) relative to normal tissue (85 +/- 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 +/- 34 [dysplasia] vs 151 +/- 44 [normal]) and 460 nm excitation (93 +/- 35 [dysplasia] vs 137 +/- 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17-40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Investigation of fiber‐optic probe designs for optical spectroscopic diagnosis of epithelial pre‐cancers

“…The human cervix was chosen as the theoretical tissue model for this study because the fluorescence efficiencies and epithelial thickness have been quantitatively characterized in previous studies (4)(5)(6)(7)(8). The thickness of the epithelial layer and that of the stromal layer were fixed at 450 μm and 2050 μm, respectively.…”

“…The thickness of the epithelial layer and that of the stromal layer were fixed at 450 μm and 2050 μm, respectively. A thickness of 450 μm was chosen for the epithelial layer to approximate the average thickness of the normal human cervical epithelium (4,6,8,32). The thickness of the stromal layer was set at 2050 μm to represent an infinitely thick tissue.…”

“…For example, fluorescence microscopy studies of transverse sections of fresh cervical tissue slices (4,5) and frozen cervical tissue blocks (6) show that there is a decrease in the intensity of collagen fluorescence in the stroma and an increase in the intensity of NADH fluorescence in the epithelium in cervical pre-cancers compared to normal tissue. Moreover, the NADH fluorescence is limited to a single layer of cells adjacent to the basement membrane in the normal epithelium, but occupies the lower one-third and two-thirds of the epithelium in low-grade and high-grade pre-cancers, respectively (5).…”

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

“…The metabolic coenzymes FAD and NADH are the primary electron acceptor and donor, respectively, in oxidative phosphorylation. Neoplastic cells have an increased metabolic demand relative to normal cells because of rapid cell division (1), and neoplastic metabolism is associated with changes in the relative concentrations of NADH and FAD (2)(3)(4). Many enzymes bind to NADH and FAD in the metabolic pathway (5), and, as favored metabolic pathways shift with cancer progression, the distribution of NADH and FAD binding sites also change (6).…”

“…A decrease in the redox ratio usually indicates increased cellular metabolic activity (8), as is typically observed in cancer cells. The redox ratio has been extensively evaluated in ex vivo normal and neoplastic tissues (2)(3)(4)9). To date, we have found no previous studies that investigated changes in the redox ratio with precancer development in three dimensions with cellular resolution in vivo.…”