## 5 Applications
### In Vivo Characterization of Biological Tissues  
TD-DOS combines distinctive features, such as non-invasiveness, deep tissue penetration, and capacity to provide functional information, which makes it a potentially powerful means for the characterization of biological tissues in vivo either for physiology studies or for clinical diagnostics. Since the initial developments of DOS, researchers have shown interest in breast cancer-related applications. They include diagnosis (detection and discrimination between malignant and benign breast lesions), as well as therapy monitoring and cancer risk assessment. Each of these goals can take advantage of a thorough optical characterization of breast tissue, as can be achieved through broadband TD-DOS spectroscopy. Breast tissue is inherently heterogeneous with two major components: fibro-glandular and adipose tissue. Inter-subject variability is very strong, spanning from fully adipose (BI-RADS category 1) to extremely dense breasts (BI-RADS category 4), where BI-RADS category is the clinical metrics to quantify \"breast density\", that is the fraction of radio-opaque fibro-glandular tissue in the breast, based on visual inspection of X-ray mammographic images [125]. Figure 13a shows typical examples of absorption spectra obtained in vivo from breasts of different types (i.e., composition), performing TD-DOS measurements in transmittance geometry, and interpreting the data with the solution of the diffusion equation for a homogeneous slab model. Notwithstanding the simple modeling, which is most often used to interpret breast data, the measured absorption properties are really sensitive to tissue composition. At short wavelengths, the major features are always the trailing edge of hemoglobin absorption, increasing upon decreasing wavelength, and the minor peak of deoxygenated hemoglobin (deoxyHb) around 760 nm. Instead, strong variability  
Figure 12: Absorption (black line) and reduced scattering (red line) spectra of thyroid tissue chromophores: (**a**) thyroglobulin, (**b**) tyrosine, (**c**) iodine, with inset depicting the anatomy of thyroid [119]. is observed above 900 nm, as a result of different tissue composition. The spectrum of the adipose breast (red symbols) is dominated by the absorption peaks of lipids around 930 and 1040 nm, while the highly fibro-glandular breast (blue symbols) is characterized by very strong water absorption around 980 nm, with collagen weakly contributing as a shoulder on the trailing edge of the water peak. Interpreting the absorption spectra with the Beer law enables the estimate of tissue composition: oxy- and deoxyhemoglobin, water lipid, and collagen (or a subset) are thus quantified. From the two forms of hemoglobin, more commonly used physiological parameters are then derived: total hemoglobin content (tHb) and oxygenation level (SO\({}_{2}\)). Figure 13b reports the reduced scattering spectra measured from the same three subjects. The spectrum of the dense (fibrous) breast typically shows a steeper slope than that of adipose or mixed breasts. That reflects the different composition: lower lipid and higher collagen content. Actually, the reduced scattering spectrum is generally interpreted with a simple empirical approximation to Mie theory [112; 113] and provides information on the microscopic structure of tissue: \(\mu_{\mathrm{s}}\)' = a\(\lambda^{\mathrm{-b}}\), where the amplitude \(a\) is related to the number of scattering centers (cell membranes, subcellular organelles, etc.) and the slope \(b\) depends on their size, being higher for smaller scattering centers. Table 1 displays the results obtained interpreting the absorption and reduced scattering spectra shown in Figure 13. Both tissue composition and scattering parameters related to the microscopic tissue structure can vary markedly with breast type. Clinical applications often impose limitations on overall measurement time. Thus, it is important to determine whether tissue quantification can be achieved even operating at a limited number of wavelengths.