For a characterization of M. ulcerans, we have designed a spectroscopy system. A prototype consisting of a standard resolution optical sensor to obtain the spectral signature of this bacterium in contact mode ( Figure 1 ) has been developed. To evaluate the potential of our approach, a prototype instrument was designed and fabricated in the LAPLACE laboratory. The instrument consists of a commercially available optoelectronic sensor combined with a fiber optic coupling system. The coupling system uses 8 surface-mounted light emitting diodes (LEDs) and a fiber optic (FOP), ensuring efficient transfer of photons over the array. The short pass filter UG11 and long pass filter GG420 are used to isolate a certain spectral range. The reflectance signals of interest are collected using a circular G11 short pass filter made of colored glass (purple color) suppressing the wavelengths below 400 nm and 700 nm. And long pass filter GG420 circular colored glass (blue color) collects reflectance signals of interest above 400 nm. This system has been developed to produce and collect diffuse reflectance models in contact imaging mode. We described the design of the instrument, presented the developed prototype and finally evaluated the performance of the prototype on tissue mimicking optical phantoms. A total of nine volunteers were recruited, including individuals with healthy skin and skin areas located near Buruli ulcer lesions, forming two categories, person A and person B. All participants were informed of the objectives and procedures of the study.

To sample the reflectance of turbid or turbid media, we used to an eight-bite spectrometer to detect and record the spectra of backscattered light on the surface of the tissue, an optical fiber, a set of LEDs, a deuterium lamp as an excitation source ( Figure 1 ). In addition, a PC was used for precise control of the acquisition of the measurements in less than five seconds and for processing the acquired data to extract the absorption and scattering properties of the tissue. Validation experiments were conducted on human subjects following the methodology used by other authors [12] [13] and using diffuse reflectance sampled from the scattering media.

No studies have been performed to obtain results on individuals based on age, gender, or ethnicity, which could be related to skin color and tissue scattering or analysis.

No subject has been excluded for the use of sunscreen, body lotion or medication, or for the presence of tattoos or skin conditions or disorders. The measurements session consisted of collecting a spectrum of the test area on the subject’s limbs and acquiring reflectance measurements of the test area. This test area was chosen for practical reasons, namely the ease of measurements with the spectrophotometer. The most intuitive approach is to place the sensor itself in contact with the tissue to collect and detect scattered photons, as proposed in [16] [17] .

To quantify possible changes in physiological parameters due to the presence of disease, a study of images of healthy skin was necessary.

Thus, the optical skin model was first validated on normal subjects. Figure 1 shows the measurements made on the left form of a healthy subject.

Then, the biological medium was described using optical properties such as the absorption coefficient µ_a, the scattering coefficient µ_s, the refractive index of the medium n and the anisotropy factor g. At the macroscopic scale, human tissues being considered as layers, we used a simplified mathematical model for each layer considered in our analysis.

For the epidermis, melanin is the main contribution, and for the dermis, the model mainly considers blood volume and blood oxygenation.

The effect of thin epidermal layer on the intensity of diffusely reflected light is based on the volume of melanin in the epidermis and the thickness of the epidermis [2] . The effective attenuation of light through the epidermis, A_epi is determined by the Beer-Lambert law, which gives:

$A_{epi}\left(\lambda \right)=\exp \left[-{\mu }_{a,epi}\left(\lambda \right)l_{epi}\right]$. (3)

where λ is the wavelength in nm, µ_a,epi the absorption coefficient of the epidermis in mm⁻¹, and l_epi the thickness of the epidermis in mm. The total absorption coefficient of the epidermis, µ_epi depends on the percentage or volume fraction of melanin in the skin and is given by:

${\mu }_{a,epi}\left(\lambda \right)=f_{mel}\cdot {\mu }_{a,mel}\left(\lambda \right)+\left(1-f_{mel}\right){\mu }_{a,skin}\left(\lambda \right)$. (4)

With f_mel the volume fraction of melanin according to skin tone, µ_a,mel the melanin absorption coefficient, and µ_a,skin the absorption coefficient of normal skin excluding base melanin. The results presented in this paper are based on the previously reported values for µ_a,mel [3] and µ_a,skin [4] , see Table 2 .‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

The remaining variable is the volume fraction fmel melanin in the epidermis. The absorption coefficient for normal skin was considered to be the same for the epidermal and dermal layers. This model does not take into account epidermal scattering because the probability of a scattering event in a 60 µm epidermis is very low, less than 5%. The absorption coefficients for melanin and dermal layer can be approximated as follows:

${\mu }_{a,mel}\left(\lambda \right)=6.6\times {10}^{11}{\lambda }^{-3.33}$ (5)

and

${\mu }_{a,skin}\left(\lambda \right)=0.244+85.3\exp \left[\frac{-\left(\lambda -154\right)}{66.2}\right]$ (6)

Gemert [5] proposed an empirical skin equation that relates the anisotropy factor g to wavelength. Epidermal anisotropy factors g_epi the dermis g_der given by:

$g_{skin}=g_{epi}=g_{der}=0.62+0.29\times {10}^{-3}\lambda$ (7)

where λ is the wavelength. The refractive indices [6] are calculated by:

$n\left(\lambda \right)=1.3199+6878{\lambda }^{-2}-1.32\times {10}^9{\lambda }^{-4}+1.11\times {10}^{14}{\lambda }^{-6}$ (8)

For the dermis, the thickest and most diffusive layer, its influence on the skin reflectance is estimated by a stochastic model of photon migration, e.g., random walk theory. Given the known random walk expression for diffuse reflectivity, R₀ semi-infinite medium with an absorbent limit and without index shift [7] :

$R_0\left(\lambda \right)=\frac{\exp \left(-2\mu \right)}{\sqrt{42\mu }}\left[1-\exp \left(-\sqrt{24\mu }\right)\right]$ (9)

where µ is the ratio of the absorption and diffusion coefficients $\frac{\mu }{{\mu }^{\prime }}$. In the case of the dermis:

$\mu =\frac{{\mu }_{dermis}}{{\mu }^{\prime }}$(10)

${\mu }_{dermis}$ and ${\mu }^{\prime }$ are the reduced diffusion and absorption coefficients of the dermis, respectively. The attenuation coefficient of this layer is related to the volume fraction of blood in the tissue and the percentage of oxygenated blood given:

$A_{dermis}\left(\lambda \right)=1.06-1.45{\left[\frac{{\mu }_{dermis}}{{\mu }^{\prime }}\right]}^{0.35}$ (11)

The dermal absorption coefficient depends on the contribution of the volume fraction of blood in the tissue, blood and the percentage of that blood that is oxygenated foxy relative fractions of HbO₂ and Hb in the blood. The dermal absorption coefficient is calculated by:

${\mu }_{a,dermis}\left(\lambda \right)=f_{blood}{\mu }_{a,blood}\left(\lambda \right)+\left(1-f_{blood}\right){\mu }_{a,skin}\left(\lambda \right)$ (12)

Where blood is the volume fraction of blood in the dermal layer and µ_a,skin is the absorption coefficient of the skin. The absorption coefficient of whole blood is expressed as:

${\mu }_{a,blood}\left(\lambda \right)=f_{blood}{\mu }_{a,oxy}\left(\lambda \right)+\left(1-f_{blood}\right){\mu }_{deoxy}\left(\lambda \right)$(13)

Data measured in vitro tend to be higher than those measured in vivo [8] . This may result from differences in sampling conditions and measurements methods used [9] .

Table 2 clearly shows that the diffusion and absorption coefficients depend on the wavelength. Based on this observation, Jacques et al. have developed an empirical equation that represents the reduced diffusion coefficient µ_s of the dermis as a function of wavelength [10] . This equation is as follows:

${\mu }_s\left(\lambda \right)=2\times {10}^5{\lambda }^{-1.5}+2\times {10}^{12}{\lambda }^{-4}$ (14)

Where the first term is Mie scattering and the second is Rayleigh scattering, λ expressed in nanometer. The optical parameters calculated at different wavelengths are presented in Table 2 . In the analysis, rather than assuming the constant anisotropy coefficient, whatever the sample analyzed, it is necessary to evaluate it according to the wavelength, this characterizes a better accuracy of the sampling step.