Fluorescence, more generally luminescence, is light not generated at high temperatures alone. It is different from incandescence, in that it usually occurs at low temperatures and is thus a form of cold body radiation. It can be caused by, for example, chemical or biochemical reactions, optical energy absorption; many kinds of luminescence are known: fluorescence, phosphorescence, bioluminescence, chemoluminescence, electroluminescence, radioluminescence, photoluminescence, and etc. Fluorescence is a property of emitting light of a longer wavelength on absorption of light energy, essentially occurs simultaneously with the excitation of a sample.
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Tattoo Carcinoma Figure 3.11 Comparison of white light (WLI) versus polarization (PI) images [33]. A freckle: polarization image removes the melanin from a freckle. A benign pigmented nevus: polarization image removes the melanin and shows apparent scatter, the drop of polarized light reflectance from epidermis lining the hair follicles is seen. Tattoo: polarization image lightens the "blackness" of the tattoo, specular reflectance of polarized light off the carbon particles yields a strong image. Malignant basal cell carcinoma: white light image underestimates the extent of the skin cancer. |
Fluorescence is characterized by the emission spectrum that is the emission obtained from a luminescent material at different wavelengths when it is excited by a narrow range of shorter wavelengths, as well as by excitation spectrum, that is, the emission spectrum monitored at one wavelength and the intensity at this wavelength is measured as a function of the exciting wavelength. Autofluorescence (AF) is a natural fluorescence of a material (a tissue) due to excitation of the endogenous fluorophores in contrast to fluorescence of a stained material (a tissues or a cell) when exogenous fluorophores are excited.
Human skin contains various types of native fluorophores with unique absorption and emission spectra (Fig. 3.6). The observations regarding the central role of the epidermal chromophores, such as keratin and NADH, in the formation of the AF spectrum of the human skin is based on a fact that the in vitro fluorescence spectra of keratin and NADH are very similar to the in vivo AF spectra of the human skin [36].
In the case of collagen and elastin, which are located predominantly within the papillary and reticular layer of dermis, the situation is a bit different. Both excitation and emission light is attenuated because of absorption by melanin. In addition, fluorescence intensity in 400-480 nm range is subject to attenuation by the other skin chromophores, such as hemoglobin, porphyrins, carotenoids etc. (Fig. 3.3). Both the total intensity and the spectral features may be affected [7,37,38].
The fluorescence intensity on excitation and emission wavelengths can best be depicted with a 3D plot (Fig. 3.12). A simple inspection of the presented spectra leads to two basic observations: the human skin exhibits a rather characteristic AF pattern, and the skin AF intensity is subject to marked individual variations.
A 2D contour plot of a 3D skin AF pattern, usually referred to as the fluorescence excitation-emission matrix (EEMs,) is shown in Fig. 3.13. One of the goals of fluorescence spectroscopy is the identification of excitation wavelengths suitable for the differentiation of various pathological conditions. Most of the biological components, which are either related
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Fluorescence intensity, a. u. Fluorescence intensity, a. u. Fluorescence intensity, a u. (a) (b) (c) Figure 3.12 3D plots of the AF spectra of the human skin measured ex vivo at different excitation wavelengths: (a) 40-year-old man; (b) and (c) 60- and 87-year-old women, respectively [7]. Measurements were performed for skin samples of 20 x 20 mm size with subcutaneous fat obtained from the patients in the course of skin plastic surgery (abdominal and lower extremities regions). |
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Figure 3.13 The excitation-emission maps (EEMs) of the in vivo human skin AF emission [39]. |
to the skin tissue structure, or are involved in metabolic and functional processes, generate fluorescence emission in the UV-visible spectral region. As a result, different morpho-func – tional conditions of the skin, related to biochemical and physiochemical alterations, can be characterized on the basis of information available in the fluorescence EMMs [7,39].