A dataset of optical spectra and clinical features acquired on human healthy skin and on skin carcinomas.

Optical spectroscopy is studied to contribute to skin cancer diagnosis. Indeed, optical spectra are modified along cancer progression and provide complementary information (e.g., on metabolism and tissue structure) to clinical examination for surgical guidance [1,2]. The current original dataset is made of autofluorescence and diffuse reflectance spectra acquired in vivo on 131 patients' skin with the SpectroLive device [3,4]. Spatially-resolved spectroscopy measurements were performed using a multi-fiber optic probe featuring 4 distances (0.4-1 mm) between excitation and collection optical fibers: spatial resolution allows spectra acquired at different distances to carry information from different depths in skin tissues. Five types of autofluorescence spectra were acquired using five different wavelength excitations (on the 365-415 nm spectral range) in order to collect information on several skin endogenous fluorophores (e.g., flavins, collagen). A sixth light source (white broadband) was used to acquire diffuse reflectance spectra carrying information about skin scattering properties and skin endogenous absorbers such as melanin and hemoglobin. Patients were proposed to be included into the clinical trial if they were suspected of suffering from actinic keratoses (precancerous skin lesions) or from basal or squamous cell carcinomas: in all cases, complete diagnostics is provided in the dataset. To increase the interest of the dataset and evaluate the dependence of optical spectra (intensity, shape) not only on pathological states but also on healthy skin features (civil age, skin age, gender, phototype, anatomical site), spectra were acquired for all 131 patients on two so-called "reference" skin sites known to rarely suffer from skin cancer: palm of the hand (featuring a thick skin type) and inner wrist (featuring thin skin). Spectra are available in .tab files: first column displays the spectral range on which intensity spectra were recorded (317-788 nm) and each following column provides an intensity spectrum acquired by each spectrometer for a given combination of light source excitation and distance. Each of the 131 folders corresponding to each of the 131 patients contains a .json file providing patients clinical features: gender, civil age, skin age, phototype score and class. All .tab files names include anatomical site and anatomopathological diagnostics of the skin site on which spectra were acquired: codes were defined to match a letter or an acronym to each diagnostic and anatomical site. To ensure quality control, a spectrum was acquired on the same calibration standard before starting spectra acquisition on each patient. It is therefore possible to follow the impact of the acquisition optical chain ageing during the 4.5 years that the patients were included. This dataset can be used by epidemiologists for the characterization of populations affected by skin cancers (gender ratio, mean age, anatomical sites typically affected, etc.); it may also be used by researchers in artificial intelligence to develop innovative methods to process such data and contribute to non-invasive diagnostics of skin cancers whose incidence is steadily increasing.

Implementation of data fusion to increase the efficiency of classification of precancerous skin states using in vivo bimodal spectroscopic technique.

This study presents the results of the classification of diffuse reflectance (DR) spectra and multiexcitation autofluorescence (AF) spectra that were collected in vivo from precancerous and benign skin lesions at three different source detector separation (SDS) values. Spectra processing pipeline consisted of dimensionality reduction, which was performed using principal component analysis (PCA), followed by classification step using such methods as support vector machine (SVM), multilayered perceptron (MLP), linear discriminant analysis (LDA), and random forest (RF). In order to increase the efficiency of lesion classification, several data fusion methods were applied to the classification results: majority voting, stacking, and manual optimization of weights. The results of the study showed that in most of cases the use of data fusion methods increased the average multiclass classification accuracy from 2% up to 4%. The highest accuracy of multiclass classification was obtained using the manual optimization of weights and reached 94.41%.

Two diagnostic criteria of optical spectroscopy for bladder tumor detection: Clinical study using 5-ALA induced fluorescence and mathematical modeling.

BACKGROUND: The study proposes to improve bladder cancer diagnosis by photodynamic diagnosis (PDD) using red-light excitation (632.8 nm) of 5-ALA induced-protoporphyrin IX. Employing 9 patients' bladders, two types of signals were used to improve diagnostic accuracy for malignancy and we also present numerical modeling of the scattering coefficient to provide biological explanation of the results obtained. METHODS: Two modalities of bladder cancer spectral diagnosis are presented: conventional PDD and intensity assessment of the diffusely reflected laser light by fiber-optic spectroscopy. Experiments are done in clinical conditions and as a series of numerical simulations. RESULTS: High-grade cancerous bladder tissues display twice a higher relative fluorescence intensity (mean value 1, n = 9) than healthy (0.39, n = 9), dysplastic (0.44, n = 5) tissues and CIS (0.39, n = 2). The laser back-scattering signal allows to discriminate most effectively high-grade cancerous and dysplastic tissues from normal. Numerical modeling of diffuse reflectance spectra reveals that spectral behavior of the back-scattered light depends on both, nuclear size and nuclear density of tumoral cells. CONCLUSIONS: Unlike the fluorescence signal, where its value is higher in the case of pathological tissues, the tendency of the laser signal to, both, decrease or increase in comparison with the signal from normal urothelium, should be perceived as a sign towards neoplasm. Numerical simulation reveals that such a double-analysis at a multiwavelength mode potentially may be used to provide diagnostic accuracy.

Clinical evaluation of a device providing simultaneous white-light and fluorescence video streams as well as panoramic imaging during fluorescence assisted-transurethral resection of bladder cancer.

The current clinical study is aimed at evaluating the clinical relevance of an innovative device (called CyPaM2 device) that for the first time provides urologists with (i) a panoramic image of the bladder inner wall within the surgery time, and with (ii) a simultaneous (bimodal) display of fluorescence and white-light video streams during the fluorescence assisted-transurethral resection of bladder cancers procedure. The clinical relevance of this CyPaM2 device was evaluated on 10 patients according to three criteria (image quality, fluorescent lesions detection relevance, and ergonomics) compared with a reference medical device. Innovative features displayed by the CyPaM2 device were evaluated without any possible comparison: (i) simultaneous bimodal display of white-light and fluorescence video streams, (ii) remote light control, and (iii) time delay for the panoramic image building. The results highlight the progress to achieve in order to obtain a fully mature device ready for commercialization and the relevance of the innovative features proposed by the CyPaM2 device confirming their interest.

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.

Spectral features selection and classification for bimodal optical spectroscopy applied to bladder cancer in vivo diagnosis.

This paper describes an experimental study combining spatially resolved autofluorescence (AF) and diffuse reflectance (DR) fibred spectroscopies to discriminate in vivo between healthy and pathological tissues in a preclinical model of bladder cancer. Then, a detailed step-by-step analysis scheme is presented for the extraction and the selection of discriminative spectral features (correlation, linear discriminant, and logistic regression analysis), and for the spectroscopic data final classification algorithms (regularized discriminant analysis and support vector machines). Significant differences between healthy, inflammatory, and tumoral tissues were obtained by selecting a reasonable number of discriminant spectral features from AF, DR, and intrinsic fluorescence spectra, leading to improved sensitivity (87%) and specificity (77%) compared to monomodality (AF or DR alone).

Classification of ultraviolet irradiated mouse skin histological stages by bimodal spectroscopy: multiple excitation autofluorescence and diffuse reflectance.

Histopathological analysis and in vivo optical spectroscopy were used to discriminate several histological stages of UV-irradiated mouse skin. At different times throughout the 30-week irradiation, autofluorescence (AF) and diffuse reflectance (DR) spectra were acquired in a bimodal approach. Then skin was sampled and processed to be classified, according to morphological criteria, into four histological categories: normal, and three types of hyperplasia (compensatory, atypical, and dysplastic). After extracting spectral characteristics, principal component analysis (data reduction) and the k-nearest neighbor classifying method were applied to compare diagnostic performances of monoexcitation AF (based on each of the seven excitation wavelengths: 360, 368, 390, 400, 410, 420, and 430 nm), multiexcitation AF (combining the seven excitation wavelengths), DR, and bimodal spectroscopies. Visible wavelengths are the most sensitive ones to discriminate compensatory from precancerous (atypical and dysplastic) states. Multiexcitation AF provides an average 6-percentage-point increased sensitivity compared to the best scores obtained with monoexcitation AF for all pairs of tissue categories. Bimodality results in a 4-percentage-point increase of specificity when discriminating the three types of hyperplasia. Thus, bimodal spectroscopy appears to be a promising tool to discriminate benign from precancerous stages; clinical investigations should be carried out to confirm these results.

Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication.

The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of GJIC have been associated with many pathological conditions, such as carcinogenesis or hereditary illness. Reliable and accurate methods for the determination of GJIC are therefore important in cell biology studies. There are several methods used successfully in numerous laboratories to measure GJIC both in vitro and in vivo. This review comments on techniques currently used to study cell-to-cell communication, either by measuring dye transfer, as in methods like microinjection, scrape loading, gap-fluorescence recovery after photobleaching (gap-FRAP), the preloading assay, and local activation of a molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation. As we will discuss in this review, these techniques are not equivalent but instead provide complementary information. We will focus on their main advantages and limitations. Although biological applications guide the choice of techniques we describe, we also review points that must be taken into consideration before using a methodology, such as the number of cells to analyze.

Mosaicing of bladder endoscopic image sequences: distortion calibration and registration algorithm.

Cancers located on the internal wall of bladders can be detected in image sequences acquired with endoscopes. The clinical diagnosis and follow-up can be facilitated by building a unique panoramic image of the bladder with the images acquired from different viewpoints. This process, called image mosaicing, consists of two steps. In the first step, consecutive images are pairwise registered to find the local transformation matrices linking geometrically consecutive images. In the second step, all images are placed in a common and global coordinate system. In this contribution, a mutual information-based similarity measure and a stochastic gradient optimization method were implemented in the registration process. However, the images have to be preprocessed in order to register the data in a robust way. Thus, a simple correction method of the distortions affecting endoscopic images is presented. After the placement of all images in the global coordinate system, the parameters of the local transformation matrices are all adjusted to improve the visual aspect of the panoramic images. Phantoms are used to evaluate the global mosaicing accuracy and the limits of the registration algorithm. The mean distances between ground truth positions in the mosaiced image range typically in 1-3 pixels. Results given for in vivo patient data illustrate the ability of the algorithm to give coherent panoramic images in the case of bladders.

Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study.

Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.