Source separation approach for the analysis of spatially resolved multiply excited autofluorescence spectra during optical clearing of ex vivo skin
Résumé
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.