WIP and WICH/WIRE modulate FAK/Src activity

5.1 Principles of Near Infrared Spectroscopy

Near Infrared Spectroscopy (NIRS) is an opto-technique, which relies on the applica-tion of the Beer-Lambert Law to measure haemoglobin concentraapplica-tions and oxygenaapplica-tion in tissue [11]. The first in vivo NIRS study has been related to Jobsis, who observed that it was possible to monitor changes in haemoglobin oxygenation and Cytochrome (Cyt aa3) reduction by applying light in the spectral range of 700-1300 nm (i.e. near-infrared) through the cats’ cranium, humans’ cranium, and dogs’ myocardium. Jobsis successfully demonstrated that the technique was able to monitor changes from nor-moxia to anoxia states from these organs. With that study, Jobsis showed the possibility of monitoring continuously and non-invasively the oxygenation states of haemoglobin, Cyt aa3, and blood volume, founding the basis for in-vivo NIRS [21].

Light interaction in tissue is composed of light absorption by chromophores and by ab-sorption and light scattering. Visible light, between 450-700 nm, is strongly attenuated due to high absorption, being unable to penetrate more than 1 cm into tissues [12].

The near-infrared region of the light spectrum is between 700 and 1100 nm and light absorption in this range is relatively low, creating a zone of relative transparency in tissues [12, 21]. In this part of the spectrum, there are three major chromophores:

oxygenated haemoglobin (HbO₂), deoxygenated haemoglobin (HHb), and reduced or oxidised forms of Cytochrome Oxidase (CtOx) [11,12,119]. As introduced in Chapter 2, haemoglobin is responsible for the transport of oxygen in blood and for the release of oxygen into tissues, whereas CtOx is an enzyme involved in the electron chain of oxygen metabolism in cells [11]. These chromophores have the peculiarity of changing their light absorption properties depending on their oxygenation state [11, 12, 119]. Each chromophore has an absorption spectrum, in which the absorption of light is expressed as a function of wavelength. The absorption spectra of HbO2, HHb, and CtOx in the near-infrared region are presented in Figure 5.1, where their different wavelength-dependent absorption characteristics in the near-infrared region are described by the curves. The isobestic point (between 800-810 nm) is the only point in the spectrum where deoxygenated haemoglobin and oxygenated haemoglobin have the same absorp-tion characteristics (i.e. extincabsorp-tion coefficient). NIRS uses the different absorpabsorp-tion properties of these chromophores to quantify their concentrations in tissues. The prin-ciple used is based on the application of the Beer-Lambert law. Although some NIRS

5.1. Principles of Near Infrared Spectroscopy

Figure 5.1: Absorption spectrum of oxygenated haemoglobin (HbO2), deoxygenated haemoglobin (Hb) and Cytochrome Oxidase (CtOx) in the near-infrared range. The absorption is expressed as specific extinction coefficient. Figure reproduced from [119].

instruments allow the additional estimation of CtOx, this chapter will mainly focus on the determination of oxygenated and deoxygenated haemoglobin only.

The Beer-Lambert law relates the absorption of light passing through a solution to the concentration of the chromophores present in the solution and the pathlength travelled by light [11,24]. Initially, Lambert described the decrease in light intensity as dependent on the distance travelled by the light beam as in Equation 5.1.

I = I0exp^−µl (5.1)

Where I₀ is the intensity of the light applied to the substance, I is the measured trans-mitted and light intensity, µ is the absorption coefficient, expressing the substance’s light absorption (in cm⁻¹), and l is the distance travelled by the light beam. Later on, Beer contributed to the concept by stating that decrease of light intensity is also due to the concentration of light absorbers in the substance. Therefore, the absorption coefficient µ can be expressed as µ = ε × c and Equation 5.1 becomes:

5.1. Principles of Near Infrared Spectroscopy

I = I₀exp^−εcl. (5.2)

Where I₀ is the intensity of the light applied to the substance, I is the measured transmitted light intensity, ε is the specific extinction coefficient (or molar absorptivity) of the substance, c is the chromophore concentration and l is the distance travelled by the light in the substance [11, 12, 17, 24]. The specific extinction coefficient ε describes the relationship between absorption properties of a substance (or chromophore) and the light wavelength (see Figure 5.1). As shown later, the extinction coefficient plays a determinant role in distinguishing two substances from each other (e.g. oxygenated and deoxygenated haemoglobin) [11,17].

By reordering Equation 5.2 and applying the logarithm, the equation can be expressed as:

A= ln^I₀ I



= ε · c · l. (5.3)

The left side of the equation represents the light attenuation A (in natural logarithm), also known as optical densities (OD), and directly relates to the concentration of the light absorbers [11]. Equation 5.3 has been reported in both natural and base 10 logarithms, but in this work the natural logarithm convention will be used.

If the substance is composed of the mixture of multiple chromophores, the addictive property of the extinction coefficients allows expressing the total light attenuation A as the linear sum of the contribution of each chromophore’s concentration as showed in Equation 5.4.

A= (ε1· c₁+ ε2· c₂+ ...εn· c_n) · l. (5.4)

This generalised equation can be also described in terms of the light wavelength λ.

A_λ = (ε1λ· c₁+ ε2λ· c₂+ ...εn_λ· c_n) · l. (5.5)

5.1. Principles of Near Infrared Spectroscopy

Where Aλ is the light attenuation at the wavelength λ, εnλ is the extinction coefficient of substance n at wavelength λ, and cnis the concentration of substance n. Equation 5.5 is an equation with n unknowns (i.e. chromophores concentrations). In order to solve this, at least n equations are required. By applying light at different wavelengths (λ₁, λ₂, ..., λn), at which the chromophore’s extinction coefficients differ, Equation 5.5 will become an exactly determined system [17].

A_λ1 = (ε_1λ1 · c₁+ ε_2λ1 · c₂+ ...εn_λ1 · c_n) · l Aλ2 = (ε_1λ2 · c₁+ ε_2λ2 · c₂+ ...εn_λ2 · c_n) · l ...

A_λn = (ε1λn· c₁+ ε2λn· c₂+ ...εn_λn · c_n) · l

(5.6)

By using multi-component analysis, the system of equations in Equation 5.6 can be solved in order to estimate the concentrations of the chromophores. This principle is used in NIRS for the estimation of the concentration of oxygenated HbO2 and de-oxygenated haemoglobin HHb. For the calculation of these two chromophores, Equa-tion 5.5 is applied at least at two wavelengths in the near-infrared region. These two wavelengths are usually selected on opposite sides with respect to the isobestic point of the haemoglobin absorption spectra (i.e. 800-810 nm). Wavelengths are often selected at both sides of the isobestic point of the spectra because HbO₂ and HHb have opposite absorption properties and, therefore, considerably different extinction coefficients ε (see Figure 5.1). A minimum of two wavelengths is required for the estimation of HbO₂and HHb, but additional wavelengths can be used, particularly if other chromophores’ con-centrations such as CtOx should be derived [11]. For the specific purpose of calculating the concentrations of HbO2 and HHb by NIRS, the system in Equation 5.6 transforms to:

A_λ1 = (εHbO_2λ1 ·[HbO2] + εHHb_λ1 ·[HHb]) · l

Aλ2 = (εHbO_2λ2 ·[HbO₂] + εHHb_λ2 ·[HHb]) · l. (5.7)

Where Aλ1 and Aλ2 are the light attenuations at wavelengths λ₁ and λ₂ respectively, ε_HbO2λ1 and εHHb_λ1 are respectively the extinction coefficients of HbO₂ and HHb at wavelength λ₁, εHbO_2λ2 and εHHb_λ2 are respectively the extinction coefficients of HbO₂

5.1. Principles of Near Infrared Spectroscopy

Figure 5.2: Effects of scattering on light propagation through a medium. In the non-scattering medium the emitted light beam with intensity I0does not undergo scattering and the detected beam I is only attenuated due to absorption. The light path length travelled by light corresponds to the thickness of the sample. A different behaviour can be noticed in the scattering medium. The light undergoes scattering in the sample, causing a prolonged light path length as well as the dispersion of some of the emitted light (i.e. P2), which does not reach the detector. Figure reproduced from [12].

and HHb at wavelength λ₂, [HbO₂] and [HHb] are the concentrations of oxygenated and deoxygenated haemoglobin, and l is the pathlength travelled by light. Equation 5.7 assumes that HbO₂ and HHb are the only chromophores present in the solution, which does not hold for tissues, where other absorbers such as lipids, melanin, and myoglobin could contribute to the overall light absorption. Later in the chapter, the approach used in NIRS to deal with this assumption will be described.

5.1.1 The Modified Beer-Lambert law

The Beer-Lambert law described so far is based on the assumption that light only undergoes absorption. However, this assumption is not valid in tissues, where near-infrared light attenuation due to scattering dominates compared to absorption (roughly 80 % scattering vs. 20 % absorption) [12, 23]. The first implication on the use of the Beer-Lambert law in presence of scattering is the impossibility of quantitatively determining the actual light attenuation, since some part of the scattered light will not reach the detector. [12]. Secondly, multiple scattering in tissue causes an increase of the pathlength travelled by light [11, 12]. Figure 5.2 visually represents both these scattering effects.

The modified Beer-Lambert law (MBLL) takes into account of the effects of scattering in tissues, by transforming Equation 5.5 in:

A = (ε₁ · c₁+ ε₂ · c₂+ ...ε · c ) · d · DP F + G. (5.8)