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We investigated pigmented skin tumour lesions in vivo and ex vivo, including benign and dysplastic nevi, as well as malignant lesions, such as pigmented basal cell carcinoma (BCC) and malignant melanoma (MM) lesions, to obtain a complex view about the feasibility of different excitation sources solely and/or in combination to induce fluorescence signal useful for diagnosis of various low-fluorescent cutaneous neoplasia. A specialized multispectral analysis of the data obtained was applied by using excitation in broad spectral range, covering ultraviolet, visible and near-infrared spectral range, that contribute considerably to: (1) fundamental determination of tumour tissues’ spectral properties, and (2) to increase the accuracy in determining the type of cutaneous pathology. The chromophores, related to the formation of ultraviolet and visible (UV-VIS) fluorescence in human normal skin and its pigmented lesions are mainly amino acids – tryptophan, tyrosine; structural proteins and their cross-links – collagen, elastin, keratin; co-enzymes - NADH, flavins; vitamins and lipids. In the near-infrared (NIR) spectral region, skin fluorescence emission properties are related to the presence of melanin pigment, lipids and endogenous porphyrins, if any, as the highest impact on the resultant emission spectrum is due to the melanin compound.

We investigated melanin-pigmented skin samples ex vivo, of benign and dysplastic nevi, as well as malignant melanoma, obtained after surgical excision, containing so called safety areas, where a normal skin could be observed, to obtain a complex and complete view about the feasibility of different excitation sources solely and/or in combination to induce fluorescence signal useful for diagnosis of pigmented cutaneous neoplasia. Using the specialized multispectral analysis of the data obtained by fluorescence using excitation in broad spectral range, covering ultraviolet, visible and near-infrared spectral range, contribute considerably to the both the fundamental determination of tumour tissues’ basic spectral parameters, and to increasing the accuracy and specificity in determining the type of pathology when spectral techniques are used that are applicable to the clinical practice. Excitation wavelengths applied namely were 365, 385, 405, 630, 785 nm. As excitation sources were used set of laser and light emitting diodes with output power in the frames of 20-50mW. Microspectrometers USB4000 and QE65000 (Ocean Optics Inc, USA) in the range of 350-1100 nm were used as detectors of the fluorescence spectra obtained using UV-VIS-NIR excitation. These are preliminary investigations based on ex vivo samples, which would be followed by in vivo investigations and comparative studies to obtain the diagnostic accuracy, using optical spectral techniques, which would surpass the existing clinical techniques for early detection and evaluation of skin cancer. The achieved results demonstrate autofluorescence spectra efficiency in highlighting biochemical changes during tumor growth in a broad spectral range.

Regretfully, because of her sudden demise, Assoc. Prof. Ekaterina Borisova is no longer amongst us. COVID-19 pulled away a brilliant scientist during the peak of her scientific career (see Fig. 1). All authors would like to express deepest condolences and sincere support to her family, friends, relatives and colleagues! We, therefore, rightfully commemorate her dedicated and devoted contribution to biophotonics, her readiness to always support, help, motivate and inspire all her colleagues and collaborators., We would like to acknowledge the support from the National Science Fund of the Bulgarian Ministry of Education and Science (NSFB-MES) under all current projects involving Assoc. Prof. Ekaterina Borisova, which are grant numbers: #DN 18-8; #КP06-N29/11/18.12.18; #KP06-N28/11/14.12.2018; #KP06-Russia-9/2020; #KP-06-N23/8; #KP-06/N38/13; #KP06-India-8/07.08.2019; #KP-06-KOST-6. Also, projects under NRRI #DO1-401/18.12.2020 and #D01-392/18.12.2020.

In the current study, an excitation laser source emitting at 785 nm (100 mW, CW) was used to obtain ex vivo fluorescence of endogenous melanin in pigmented skin with benign, dysplastic and malignant cutaneous lesions. The samples were obtained after surgical removal during standard excision procedure and split for spectral analysis and histological verification. The samples of benign (BN-5) and dysplastic (DN-3) nevi and of malignant melanoma (MM-7) were used as representative for harmless/harmful cutaneous neoplasia with a similar melanin pigmentation. Emission in the range 800 – 1100 nm was detected and compared using a USB4000 micro-spectrometer (Ocean Optics Inc., USA). The photobleaching dynamics was observed for the emission maxima at 825 nm for 10 minutes with a step of 20 s. These near-IR autofluorescence spectra may be assigned to a single fluorophore if appropriate excitation wavelength is applied, which simplifies the rapid analysis related to the appearance and concentration evaluation of a given type of endogenous fluorophore, as well as allows the evaluation of some parameters, such as photobleaching dynamics, as a diagnostic indicator assessing the tissue state. A diagnostic accuracy of 93.3% was achieved for MM lesions validation when the NIR fluorescence intensity and photobleaching rate values were used to discriminate between nevi and melanoma lesions.