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While EEG is very powerful in detecting brief cognitive processes in the range of milliseconds, fNIRS provides a better localization of these processes. Compared to fMRI, the fNIRS can be used to monitor not only reduced hemoglobin, but also oxygenated and total hemoglobin, as well as reflecting the changes in local blood volume (Wallois et al., 2012).

fNIRS measures hemodynamic oxygenation changes by detecting changes in emitted light in- tensity after passage through a tissue in response to functional stimulation (Obrig & Villringer,

60 2003; Villringer & Chance, 1997). The measurement is based upon three physiological condi- tions: biological tissue is relatively transparent for light within the range from 600 to 1000 nm (infrared light). Oxygenated and deoxygenated hemoglobin (oxy-Hb, deoxy-Hb) have charac- teristic absorption properties in this spectral range and changes in the cerebral tissue lead only to minor variations in scattering (Obrig, 2002). The near-infrared light can penetrate to a sig- nificant depth in the cortical tissue (i.e., 1 to 2 cm) without being extensively absorbed by back- ground tissue constitutes (Obrig & Villringer, 2003; Wallois et al., 2012). However, light below 650 nm is too strongly absorbed by hemoglobin and above 950 nm too strongly by water. Thus, the spectral range of the “optimal window” is found at around 800 nm due to oxy-Hb and deoxy- Hb having different maxima and minima in their absorption spectra (Scholkmann et al., 2014). Light and biological tissue interact primarily in two means: absorbtion and scattering. Hemo- globin functions as chromophores (c) which absorb, reflect and scatter light that is thus attenu- ated (light attenuation A). In the near-infrared light spectrum, only a small portion of light is absorbed by the hemoglobin while the majority of it leaves the tissue and is detected by light emitters. The difference between the emitted and the detected light intensities is determined by the oxygenation of the hemoglobin and thus indirectly reflects brain activity (Obrig, 2002; Wal- lois et al., 2012).

Each chromophore has a typical spectrum of absorption, which what means that oxygenation changes lead to a change in tissue absorption at different wave lengths (Obrig, 2002). Secondary to changes in brain activity, the concentration of oxy-Hb and deoxy-Hb varies. Hence, the amount of detected light changes depending on the absorption in the activated brain region (absorption coefficient µa) and the time required for the non-absorbed light to be emitted from the cortical tissue after different events of scattering (scattering coefficient µs). The different spectra of absorption enables the differentiation of the oxygenation changes in oxy-Hb and deoxy-Hb (i.e., hemoglobin has different extinction rates depending on the oxygenation status;

61 Buckow, 2008; Obrig, 2002; Obrig & Villringer, 2003). These changes are similar to differ- ences in the color of arterial and venous blood (Rossi et al., 2011). The variations in the two chromophores can be calculated from changes in optical densities by using the modified Lam- bert-Beer-Law. It is necessary to use the modified law, as it compensates for the highly light scattering nature of biological tissue that makes the quantitative measurement of light changes impossible (Cope & Delpy, 1988). The original Lambert-Beer-Law was first discovered by Bouguer in 1729 (Bouguer, 1729) and is often attributed to Lambert, although he cited Bouguer´s work (Lambert, 1760). The law was extended by Beer to quantify concentrations (Beer, 1852). The law describes the relation between the light attenuation (A), which is optically characterized by the absorption (µa) and scattering (µs) coefficient, and the concentration of chromophores/absorber (c) in the cuvette model (see Fig. 5). In an idealistic cuvette model with the assumption that no scattering takes place (µs = 0; with endless dilute solutions), the con- centration of a substance can be determined by the absorption coefficient (Fig. 5, photon 2). With the assumption of µs = 0, the absolute concentration of chromophores can be calculated with the Lambert-Beer-Law (Buckow, 2008; Obrig, 2002):

� = log � = � ∗ ∗  ₌ �

�∗ with: µa = ɛ * c; µs = 0

with:

A = light attenuation; Io = emitted light intensity; I = detected light intensity; ɛ = extinction coefficient in mol/l-1 _cm-1 _{(i.e., specific absorption of the chromophores); c = concentration of}

the chromophores (absorber) in mol/l; d = geometric distance (cuvette width). In the original Lambert-Beer-Law the extinction of the light (Io/I) is proportional to the concentration (c) of the chromophores multiplied by the constant extinction coefficient (ɛ) for the particular ab- sorber and the distance (d) corresponding to the cuvette width. This law holds as long as photons

62 are either transmitted in a straight line directly to the detector (Fig. 5, photon 1) or absorbed (Fig. 5, photon 2) (Villringer & Chance, 1997).

However, in biological tissue, absorption and scattering cannot be separated (Obrig, 2002). Thus, the Lambert-Beer-Law is only valid in non-scattering media and cannot be applied to biological tissue (Scholkmann et al., 2014). To calculate the changes of µa in biological tissue, the highly scattering nature of biological tissue must be therefore considered. This scattering is mostly caused by membranes, cells, and boundary surfaces between tissue layers (Buckow, 2008). Nevertheless, to identify concentration changes of the chromophores, a constant scatter- ing is assumed (Cope & Delpy, 1988). This assumption is based on the fact that the scattering in the tissue is indeed high but constant over the time of measuring. In other words, it is assumed that the biological tissue has background properties (fixed absorbers such as melanin, water, fat and, therefore, constant scattering) which influences the light attenuation, but that changes of

µa occur only through dynamic chromophores (e.g., oxy-Hb and deoxy-Hb). Thus, changes in

the cerebral blood flow, which is marked by concentration changes in oxy-Hb and deoxy-Hb, influences the absorption coefficient of the tissue more than the scattering coefficient (domi- nated by cell density) (Obrig, 2002). Under this assumption, concentration changes can be cal- culated with the modified Lambert-Beer-Law. The modified formula, which is needed for higher substance concentrations and higher scattered mediums, considers the longer path length of the light (see Fig. 5, photon 3) and the loss of light (see Fig. 5, photon 5) due to scattering (Buckow, 2008; Delpy et al., 1988; Villringer & Chance, 1997):

� = � ∗ ∗ ∗ �� +

The modified Lambert-Beer-Law was therefore completed with a term G, which is a measure of the signal loss and a term DPF, which accounts for the longer path length than the original geometric emitter-detector-distance due to light scattering (Villringer & Chance, 1997). The

63 term G and DPF is assumed to be constant over the time of measuring (Buckow, 2008). The term G is dependent on the geometry and unknown factors, which makes the determination of the absolute chromophores concentration not possible. The term G can be neglected when the deviation over the time is considered (ΔA(Δt,λ) = A(t1,λ ) - A(t0,λ)), under the assumption that the emitted light intensity remains constant (Io). If different chromophores contribute to the light attenuation than the total attenuation must include the part attenuation of the absorbed chromophores. Thus, the formula for a specific wavelength (λ) is as follows (Buckow, 2008; Villringer & Chance, 1997):

∆� λ = ∑ [�� � λ ∗ ∆ � ∗ �� λ + d]

A, ɛ, and DPF depend on the wavelength λ. The index i indicates the particular chromophore (e.g., oxy-Hb or deoxy-Hb). If discrete wavelengths are used, the number of wavelengths cor- responds with the number of chromophores.

The converted formula to determine the concentration changes in oxy-Hb and deoxy-Hb is as follows (Scholkmann et al., 2014):

∆ [� − �] = ∗ � [� − �]λ ∗ �[ � − �]λ ∗ ∆� λ �� λ ∆ [ � − �] = ∗ � [� − �]λ ∗ �[ � − �]λ ∗ ∆� λ �� λ

However, besides determining the oxy- Hb and deoxy-Hb, the total hemoglobin (tot-Hb) can be calculated: tot-Hb = oxy-Hb + deoxy-Hb. If a brain region is activated, deoxy-Hb decreases

64 while oxy-Hb increases. In the most cases, this results in an increase of tot-Hb which represents the corpuscular blood volume (Obrig, 2002).

Fig. 5. Absorption and scattering in the cuvette model. The attenuation of the light is measured between the light

source (Io) and the detector (Ix). The light flies through a medium with an optical density (OD), which is charac- terized by the absorption (µa) and scattering (µs) coefficient, in a cuvette with a certain width (d). The original Beer-Lambert-Law holds as long as a photon (numbered arrows) is either transmitted in a straight line directly to the detector (photon 1) or absorbed (photon 2) under the requirement of endless dilute solutions (µs =0). The concentration of an absorber (c) can be thus calculated through the light attenuation. However, an absorber can be located in the shadow of another absorber, resulting in the concentration being too low. In biological tissue with higher substance concentrations and significant light scattering, the original Beer-Lambert-Law cannot determine of whether a photon was absorbent (photon 2), scattering (photon 3), or both (photon 4). Further, it cannot calculate of whether the light is transmitted in a straight line directly to the detector (photon 1), if it is lost (photon 5), or has a longer path length (photon 3) due to light scattering. Thus, the formula must be modified, as described in the text (on the basis of Buckow, 2008; Obrig, 2002; Villringer & Chance, 1997).

65 The modified Lambert-Beer-Law serves only in continuous wave approaches (CW; i.e., without time resolution) for quantifying chromophore concentrations (which was used for the present dissertation). The term CW means that the instrument is based on a light intensity measurement (i.e., light is emitted into the tissue and the intensity of the re-emerging light is measured). In contrast, time resolved techniques additionally measures the time of flight (i.e., the time that the light needs to travel through the tissue). The disadvantage of CW systems is that the optical properties of tissue (i.e., µs and µa) cannot be fully determined and therefore oxy-Hb and de- oxy-Hb cannot be determined absolutely (Scholkmann et al., 2014). However, CW systems can provide a measurement of relative concentrations changes of oxy-Hb and deoxy-Hb in the time course, such as between cognitive activation and a baseline condition (Fallgatter et al., 2004). In general, the fNIRS allows only a “blurry image” of activated brain regions and is therefore not as accurate as in the fMRI. However, this disadvantage can be acceptable secondary to the rather undemanding technical requirements of fNIRS and a high specificity for the parameters monitored. Thus, compared to the fMRI, the fNIRS permits a better quantification of the con- centration changes of chromophores (Obrig, 2002; Obrig & Villringer, 2003).