Fluorescence intensity is a function of both the excitation and emission wavelength in samples containing multiple fluorophores such as human tissue. Tissue autofluorescence originates from structural proteins (collagen and elastin) and their cross-links, metabolic co-factors (NAD(P)H and FAD), aromatic amino acids (tryptophan, tyrosine and phenylalanine), and porphyrins, each of which have a characteristic excitation spectrum with an associated characteristic emission. Collagen and elastin are major parts of the extracellular matrix and predominantly found in stromal tissue layers. Nicotine adenine dinucleotide (NAD(P)) and flavin adenine dinucleotide (FAD) are reduced in the citric acid cycle (anaerobic glycolysis) to FADH and NAD(P)H, which are utilized as coenzymes in the electron transport chain (aerobic glycolysis). Because the pyridine nucleotides and flavins play an integral role in cellular metabolism, it is possible to assess the metabolic status of tissue by monitoring changes in the concentrations of these electron carriers. Increased metabolism will result in increased NAD(P)H and decreased FAD concentrations. Recent studies in the cervix indicate that fluorescent components in the stroma change before cells invade and that optical probing of the stroma may be an important indicator of a premalignant process that is still confined to the epithelium.

Light undergoes elastic scattering and potentially absorption when traveling inside the tissue (Fig A2). Light remitted from the tissue surface is known as diffuse reflectance. Scattering depends on small-scale fluctuations of refractive indices which originate from cell organelles such as chromatin, mitochondria and membrane. Since absorption in tissue is largely due to hemoglobin and because oxygenated and deoxygenated hemoglobin absorb light differently, average blood oxygenation can be determined from reflectance spectra. Several groups have shown that tissue backscattering is altered by the intracellular micro structure such as nuclear size and