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

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

Inversion de modèle et séparation de signaux de spectroscopie optique pour la caractérisation in vivo de tissus cutanés

The detection of pre-cancerous biological tissues during clinical diagnosis is made possible by the development of optical spectroscopic methods, also known as optical biopsy. These methods make it possible to identify changes in the metabolism and structure of biological tissues. In this thesis, optical measurements are performed on skin tissues using an optical device that integrates two modalities: spatially resolved diffuse reflectance and autofluorescence. Spectra exploitation involves solving a non-linear inverse problem to estimate the optical properties using a light-tissue interaction model. From these optical properties, it is possible to characterize the condition of the tissue, healthy or abnormal. The first contribution of the thesis is to propose an inversion method that integrates a fast Monte Carlo model and a cost function managing two spectral modalities in order to improve the accuracy of the estimation of optical properties, regardless of the geometry of the probe. The second contribution is to study the kinetics of fluorophores present in tissues by a source separation approach, which does not require the use of a Monte Carlo model. In this approach, a collection of spectra acquired over time is analyzed together, with few a priori assumptions about the shape of the source signals. The sensitivity of this approach is validated by the use of optical clearing that increase tissue transparency by decreasing photon diffusion and absorption in tissues, thus providing access to more accurate in-depth information.La détection de tissus biologiques pré-cancéreux au cours d’un diagnostic clinique est rendue possible grâce au développement de méthodes spectroscopiques optiques, appelées également biopsie optique. Ces méthodes permettent de mettre en évidence les modifications du métabolisme et de la structure des tissus biologiques. Dans cette thèse, les mesures optiques sont réalisées sur les tissus cutanés à l’aide d’un instrument intégrant deux modalités : la réflectance diffuse résolue spatialement et l’autofluorescence. L’exploitation des spectres consiste à résoudre un problème inverse non-linéaire pour estimer les propriétés optiques du milieu sondé à l’aide d’un modèle d’interaction lumière-tissu. À partir de ces propriétés optiques, la caractérisation de l’état du tissu, sain ou anormal, est possible. La première contribution de la thèse est de proposer une méthode d'inversion qui intègre une simulation Monte Carlo rapide et une fonction coût gérant les deux modalités spectrales dans le but d’améliorer la précision de l’estimation des propriétés optiques, quelle que soit la géométrie de la sonde. La deuxième contribution de la thèse est d'étudier la cinétique des fluorophores présents dans les tissus par une approche de séparation de sources, qui ne nécessite pas le recours à une simulation Monte Carlo. Dans cette approche, une collection de spectres acquis au cours du temps est analysée conjointement, avec peu d'hypothèses a priori de la forme des signaux sources. La sensibilité de cette approche est validée par l’emploi d’agents de clarification optique qui augmentent la transparence des tissus en diminuant la diffusion et l’absorption des photons dans les tissus, et donnent ainsi accès à des informations plus précises en profondeur

Model inversion and separation of optical spectroscopy signals for in vivo characterization of skin

La détection de tissus biologiques pré-cancéreux au cours d’un diagnostic clinique est rendue possible grâce au développement de méthodes spectroscopiques optiques, appelées également biopsie optique. Ces méthodes permettent de mettre en évidence les modifications du métabolisme et de la structure des tissus biologiques. Dans cette thèse, les mesures optiques sont réalisées sur les tissus cutanés à l’aide d’un instrument intégrant deux modalités : la réflectance diffuse résolue spatialement et l’autofluorescence. L’exploitation des spectres consiste à résoudre un problème inverse non-linéaire pour estimer les propriétés optiques du milieu sondé à l’aide d’un modèle d’interaction lumière-tissu. À partir de ces propriétés optiques, la caractérisation de l’état du tissu, sain ou anormal, est possible. La première contribution de la thèse est de proposer une méthode d'inversion qui intègre une simulation Monte Carlo rapide et une fonction coût gérant les deux modalités spectrales dans le but d’améliorer la précision de l’estimation des propriétés optiques, quelle que soit la géométrie de la sonde. La deuxième contribution de la thèse est d'étudier la cinétique des fluorophores présents dans les tissus par une approche de séparation de sources, qui ne nécessite pas le recours à une simulation Monte Carlo. Dans cette approche, une collection de spectres acquis au cours du temps est analysée conjointement, avec peu d'hypothèses a priori de la forme des signaux sources. La sensibilité de cette approche est validée par l’emploi d’agents de clarification optique qui augmentent la transparence des tissus en diminuant la diffusion et l’absorption des photons dans les tissus, et donnent ainsi accès à des informations plus précises en profondeur.The detection of pre-cancerous biological tissues during clinical diagnosis is made possible by the development of optical spectroscopic methods, also known as optical biopsy. These methods make it possible to identify changes in the metabolism and structure of biological tissues. In this thesis, optical measurements are performed on skin tissues using an optical device that integrates two modalities: spatially resolved diffuse reflectance and autofluorescence. Spectra exploitation involves solving a non-linear inverse problem to estimate the optical properties using a light-tissue interaction model. From these optical properties, it is possible to characterize the condition of the tissue, healthy or abnormal. The first contribution of the thesis is to propose an inversion method that integrates a fast Monte Carlo model and a cost function managing two spectral modalities in order to improve the accuracy of the estimation of optical properties, regardless of the geometry of the probe. The second contribution is to study the kinetics of fluorophores present in tissues by a source separation approach, which does not require the use of a Monte Carlo model. In this approach, a collection of spectra acquired over time is analyzed together, with few a priori assumptions about the shape of the source signals. The sensitivity of this approach is validated by the use of optical clearing that increase tissue transparency by decreasing photon diffusion and absorption in tissues, thus providing access to more accurate in-depth information

Influence of cost functions and optimization methods on solving the inverse problem in spatially resolved diffuse reflectance spectroscopy

Published in Proc. SPIE 10063, Dynamics and Fluctuations in Biomedical Photonics XIV, 100630YInternational audienceDiffuse reflectance spectroscopy (DRS) has been acknowledged as a valuable optical biopsy tool for in vivo characterizing pathological modifications in epithelial tissues such as cancer. In spatially resolved DRS, accurate and robust estimation of the optical parameters (OP) of biological tissues is a major challenge due to the complexity of the physical models. Solving this inverse problem requires to consider 3 components: the forward model, the cost function, and the optimization algorithm. This paper presents a comparative numerical study of the performances in estimating OP depending on the choice made for each of the latter components. Mono- and bi-layer tissue models are considered. Monowavelength (scalar) absorption and scattering coefficients are estimated. As a forward model, diffusion approximation analytical solutions with and without noise are implemented. Several cost functions are evaluated possibly including normalized data terms. Two local optimization methods, Levenberg-Marquardt and TrustRegion-Reflective, are considered. Because they may be sensitive to the initial setting, a global optimization approach is proposed to improve the estimation accuracy. This algorithm is based on repeated calls to the above-mentioned local methods, with initial parameters randomly sampled. Two global optimization methods, Genetic Algorithm (GA) and Particle Swarm Optimization (PSO), are also implemented. Estimation performances are evaluated in terms of relative errors between the ground truth and the estimated values for each set of unknown OP. The combination between the number of variables to be estimated, the nature of the forward model, the cost function to be minimized and the optimization method are discussed

Caractérisation par spectroscopie optique des classes tissulaires rencontrées lors de résection chirurgicale des carcinomes cutanés

National audienceLes cancers cutanés non mélanocytaires (appelés carcinomes cutanés) sont les cancers les plusfréquents, et leur prévalence est en augmentation dans l’hémisphère Nord : 20 % des plus de 60 ans sontporteurs de lésions précancéreuses [1]. La chirurgie est le traitement de référence dans la prise en chargethérapeutique de ces cancers et le plus souvent, la résection chirurgicale se fait en « fuseau » (Fig. 1). Cefuseau contient 3 types de tissu cutané dont la nature est évaluée visuellement par le chirurgien (avantdiagnostic définitif par anatomo-pathologie) : lésionnel (« L », partie visible du carcinome cutané), périlésionnel(« PL », marge de sécurité) et non-lésionnel (« NL », extrémités du fuseau).L’essai clinique SpectroLive mené actuellement au CHR Metz-Thionville propose d’évaluer lacapacité de la spectroscopie optique résolue spatialement à caractériser la nature histologique et photobiologiquede ces 3 types de tissus. Le dispositif SpectroLive [2] permet l’acquisition de spectres d’autofluorescence(pics d’excitation entre 365 et 415 nm) et de réflectance diffuse recueillis à 4 distances interfibres: de 0,4 à 1 mm. Le but est d’aider le chirurgien dans son orientation diagnostique visuelle pourdiscriminer plusieurs classes tissulaires d’intérêt clinique : (i) sain, (ii) cancer, (iii) kératose actinique àhaut potentiel évolutif et (iv) kératose à faible potentiel évolutif. Afin d’augmenter le rapport signal àbruit, 3 spectres sont acquis consécutivement sur un même site, pour chacun des 3 types de tissusrencontrés au sein du fuseau chirurgical (L, PL et NL). Les résultats préliminaires montrent unediminution globale du signal entre cancer (site de mesure « L ») et tissu sain (site de mesure « NL »), enRD à toutes les distances inter-fibres (Fig. 1b) comme en auto-fluorescence

Analysis of skin intrinsic fluorophore contributions during optical clearing: source separation technique applied to spatially resolved multiply excited autofluorescence spectra

International audienc

Impact of standardization of autofluorescence and diffuse reflectance spectra on diagnosis accuracy of optical spectroscopy used for skin carcinomas diagnosis

International audienc

Human skin optical property modifications upon optical clearing agents estimated from spatially-resolved tissue optical spectroscopy

International audienceOptical clearing agents’ impact on skin optical properties (OP) was investigated using spatially – resolved bimodal spectroscopy and inverse Monte Carlo modelling. Optical spectra were obtained on an original hybrid model made of ex vivo human skin as a top layer and of agarose and porphyrin based – nanoparticles as a bottom layer. First results are given in terms of estimated absorption and scattering coefficients of skin layer before and after OCA application

Spatially resolved spectroscopy for guiding margin delineation during human skin carcinomas resection: first clinical results on diffuse reflectance and autofluorescence spectra and in vivo skin optical properties

Abstract published in Proc. SPIE 10685, Biophotonics: Photonic Solutions for Better Health Care VI, 106851J (17 May 2018)International audienceOptical spectra acquired on skin depend greatly on skin content in chromophores especially on photons absorbers (melanin) and on fluorescent molecules such as collagen and elastin. Such skin content in chromophores varies from one person to another one even within the same phototype class. Therefore, optical spectra must be standardized in order to be as independent as possible of inter-individual variability. Such a standardization should help increase the diagnosis accuracy of optical spectroscopy. In this study, we aim at defining the anatomical site that would allow best standardization. Standardization is evaluated through the ability of spectroscopy to discriminate pairs of histological classes such as BCC, SCC, AK and normal skin. Bimodal spectroscopy is used combining AutoFluorescence (AF) and Diffuse Reflectance (DR). Three anatomical sites are compared for reference spectra acquisition: non-lesional (NL) skin sites (i.e. the same anatomical sites as for BCC, SCC or AK), inner hand and inner wrist. AF raw data show a high (40 %) inter-individual variability of reference spectra intensity acquired on any of the three anatomical sites. Standardization using reference spectra allow best discrimination of histological classes when reference spectra acquired on NL skin sites and on inner hand are used for standardization of AF spectroscopy and DR spectroscopy, respectively