Abstract
Spatially resolved multiply excited autofluorescence spectroscopy is a valuable optical biopsy technique to investigate skin UV-visible optical properties in vivo in clinics. However, it provides bulk fluorescence signals from which the individual endogenous fluorophore contributions need to be disentangled. Skin optical clearing allows for increasing tissue transparency, thus providing access to more accurate in-depth information. The aim of the present contribution was to study the time changes in skin spatially resolved and multiply excited autofluorescence spectra during skin optical clearing. The latter spectra were acquired on an ex vivo human skin strip lying on a fluorescent gel substrate during 37 minutes of the optical clearing process of a topically applied sucrose-based solution. A Non Negative Matrix Factorization-based blind source separation approach was proposed to unmix skin tissue intrinsic fluorophore contributions and to analyze the time evolution of this mixing throughout the optical clearing process. This spectral unmixing exploited the multidimensionality of the acquired data, i.e., spectra resolved in five excitation wavelengths, four source-to-detector separations, and eight measurement times. Best fitting results between experimental and estimated spectra were obtained for optimal numbers of 3 and 4 sources. These estimated spectral sources exhibited common identifiable shapes of fluorescence emission spectra related to the fluorescent gel substrate and to known skin intrinsic fluorophores matching namely dermis collagen/elastin and epidermis flavins. The time analysis of the fluorophore contributions allowed us to highlight how the clearing process towards the deepest skin layers impacts skin autofluorescence through time, namely with a strongest contribution to the bulk autofluorescence signal of dermis collagen (respectively epidermis flavins) fluorescence at shortest (respectively longest) excitation wavelengths and longest (respectively shortest) source-to-detector separations.
Highlights
Multimodal fibered optical spectroscopy is a point optical biopsy technique combining Spatially Resolved (SR) diffuse reflectance and AutoFluorescence (AF) intensity spectra measurements
The spectral sources Sestimated by the Alternating Least Squares (ALS)-based Negative Matrix Factorization (NMF) method, according to all Source-to-Detector Separations (SDS) D1:4, all fluorescence excitation wavelengths λ1e:x5c and all time points during the Optical Clearing process t1:8, are shown in Fig. 4 for both numbers of sources NS = 3 and NS = 4
Resolved (SR) multiply excited AutoFluorescence (AF) spectra were acquired on an ex vivo skin strip lying on a fluorescent gel substrate
Summary
Multimodal fibered optical spectroscopy is a point optical biopsy technique combining Spatially Resolved (SR) diffuse reflectance and AutoFluorescence (AF) intensity spectra measurements. Multiple excitation light induced AF spectroscopy applied on skin provides global information about the composition and metabolism of the cutaneous tissue in probing the intrinsic fluorophores contained in cells (keratin, reduced forms of pyridine nucleotides NAD(P)H, Flavin Adenine Dinucleotide FAD, porphyrins) and in extracellular matrix (elastin, collagen) [4, 5]. The use of several excitation wavelength sources allows one for scanning the relative contributions of various fluorophores according to their respective excitation and emission spectra. The latter are involved in the shape formation of the global AF spectra collected by spectroscopy which depends on the penetration depths of the different excitation wavelengths and on differences in influence of pigments, especially hemoglobin [9]. The spectral shape of skin global AF signals is modified in accordance with the concentration of fluorophores, which is correlated with the biochemical activity related to skin pathological states
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