Microanatomical and Biochemical Origins of Normal and Precancerous Cervical Autofluorescence Using Laser-scanning Fluorescence Confocal Microscopy ¶

Abstract

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 mm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower twothirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.

Design and characterization of a novel multimodal fiber-optic probe and spectroscopy system for skin cancer applications

“…LIFS is bio-chemically sensitive as it interrogates endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and collagen. Their fluorescence levels change with neoplastic progression that is associated with altered cellular metabolic pathways (NADH, FAD) 26 or an altered structural tissue matrix (collagen). [26][27][28][29] Raman spectroscopy exploits the inelastic scattering (socalled "Raman" scattering) phenomena to detect spectral signatures of important disease progression biomarkers, including lipids, proteins, and amino acids.…”

“…Their fluorescence levels change with neoplastic progression that is associated with altered cellular metabolic pathways (NADH, FAD) 26 or an altered structural tissue matrix (collagen). [26][27][28][29] Raman spectroscopy exploits the inelastic scattering (socalled "Raman" scattering) phenomena to detect spectral signatures of important disease progression biomarkers, including lipids, proteins, and amino acids. Raman spectroscopy is more constituent-specific than fluorescence and is capable of spectrally "breaking down" the biochemical composition; however, the two techniques are complementary as they probe different bio-molecular species.…”

Autofluorescence Imaging and Spectroscopy of Human Lung Cancer

“…This phenomenon could be due to the increased vasculature around cancerous lesions, and partially due to changes in the fluorescent properties of the tissues. Previous studies have also been performed on blood samples [26], urine samples [27], animal models [28], cell lines [29], and human cervical tissue sections [30,31] based on the fluorescence spectroscopy. Direct studies on the optical properties of lung tissues may be particularly useful for intraoperative margin assessment during tumor resection [32].…”

Autofluorescence and diffuse reflectance spectroscopy of oral epithelial tissue using a depth-sensitive fiber-optic probe

“…While fluorescence microscopy studies of excised fresh tissue provide insight into the spatial dependence of optical property changes that accompany neoplasia [17,18], it is more difficult to interpret spectroscopic measurements made from intact tissue. The precise mechanisms underlying the observed changes in tissue autofluorescence are not well understood [19].…”