Natural tissue autofluorescence (AF) originates primarily from specific endogenous fluorophores located in mitochondria (NAD(P)H and flavin coenzymes, porphyrins, and lipopigments), structural proteins in extracellular matrix (ECM; collagen and elastin), cell lysosomes (lipofuscins), and various aromatic amino acids appearing in many proteins within the living body. Amino acid tryptophan (Trp) with two absorption maxima at 220 and 287 nm, both emitting at 350 nm, is one of the most abundant protein fluorophores but, due to lack of specificity, is seldom used for biomedical diagnostics purpose. e major endogenous fluorophores excited above 300 nm are structural proteins such as collagen, elastin, or keratin; metabolites; and enzyme cofactors, namely, cellular flavins and reduced nicotinamide adenine dinucleotide (phosphate), also known as NAD(P)H. Reduced NAD(P)H is one of the most studied endogenous fluorophores, with excitation maximum at 350-380 nm and emission near 440 nm. Oxidized flavins are, on the other hand, excited near 450 nm with emission maximum near 515 nm. Other endogenous fluorescence compounds include lipofuscins, age-related lipophilic materials with wide excitation and emission bands spanning over the visible spectral range. Important sources of endogenous fluorescence also include pigments (such as melanin or bilirubin, porphyrins, as well as different vitamins and pterins). Table 3.1 summarizes excitation, emission, and lifetime characteristics of endogenous fluorophores found in biological systems, while Table 3.2 enumerates fluorescence parameters of known endogenous fluorophores in different cells and tissues. Molecular structure of the representative fluorophores and optically active molecules found in tissues and discussed further in this chapter is shown in Figure 3.1, while their excitation and emission spectra are illustrated in Figure 3.2.