Non-invasive method for detecting, visualizing and / or quantifying endogenous fluorophores, such as melanin, in biological tissues

Abstract

A method for detecting, quantifying and / or visualizing endogenous fluorophores, such as melanin, in a biological tissue, the method comprising: acquiring a plurality of two-dimensional (2D) multiphoton images of fluorescence intensity, or combined multiphoton FLIM images, each image being acquired in a plane substantially perpendicular to a surface of the biological tissue, the image is processed to detect boundaries of the surface of the biological tissue and to detect endogenous fluorophores, and information representative of a distribution of endogenous fluorophores in the biological tissue relative to the boundaries is generated based on the processed image, in particular as a function of the distance from the boundaries.

Description

Field of the Invention

[0001] The present invention relates to the observation of biological tissues, particularly keratinous materials such as skin.

[0002] The present invention more particularly but not exclusively relates to methods aimed at determining the distribution of melanin in biological tissues, particularly for the purpose of evaluating the effect of cosmetic treatments thereon.

[0003] Background

[0004] The color of some biological tissues is closely related to the amount and three-dimensional distribution of melanin. Nowadays, different methods can be used to characterize the amount and distribution of melanin in vitro or in vivo.

[0005] Currently, the gold standard method for melanin quantification in the skin is the chemical analysis by high performance liquid chromatography (HPLC) of melanin degradation products. Although highly specific, it requires the degradation of ex vivo samples and does not provide information on the epidermal distribution of melanin. The Fontana-Mason staining of horizontal skin sections allows insight into the 2D melanin distribution and content, but has the drawback of non-specific stratum corneum (SC) staining, while the Warthin-Starry staining can provide more sensitive and specific melanin detection. Transmission electron microscopy enables the analysis of the melanosome pattern within epidermal cells and its variations according to skin phenotypes.

[0006] For many years, different techniques have been investigated to provide the possibility of non-invasive melanin detection based on the optical properties of melanin, broadband ultraviolet and visible light absorption, fluorescence emission spectra and lifetimes. Spontaneous Raman spectroscopy has found that pheomelanin has specific peaks within the "silent region" of the Raman spectrum, thus providing a direct way for the specific non-invasive detection of 3D pheomelanin in skin samples by CARS (Coherent Anti-Stokes Raman Scattering) imaging. Recently, the combined analysis of specific Raman bands together with the spontaneous fluorescence of the skin excited by near-infrared single photons has been used to estimate the xz depth profile of the in vivo melanin fraction, but this method lacks the spatial localization of intracellular melanin and cannot image the entire epidermis. Pump-probe imaging has also been shown to be valuable in analyzing melanin in 2D thin skin sections of pigmented lesions (i.e., for its eumelanin / pheomelanin discrimination).

[0007] In 1979, melanin imaging based on its endogenous fluorescence was demonstrated on human skin sections by conventional single-photon excitation fluorescence microscopy, and 20 years later, melanin imaging based on its endogenous fluorescence was demonstrated in vivo on the forearm skin using two-photon excitation / multiphoton microscopy.

[0008] Multiphoton imaging enables three-dimensional characterization of biological tissues with sub-micron resolution. The imaging depth varies according to the tissue, and for human skin, the imaging depth can be approximately 200 μm. Multiphoton microscopy provides non-invasive label-free 3D visualization of the epidermal layer and the epidermal distribution of melanin by utilizing endogenous autofluorescence signals from keratin, NAD(P)H and FAD metabolic coenzymes, and melanin.

[0009] Under multiphoton excitation at 760 nm, intensity-based methods for melanin detection have been proposed, which are performed in vitro on reconstructed pigmented epidermis, such as in FR 2944425; and in vivo on human skin, such as in Ait ElMadani, H. et al. “In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation” J Biomed Opt 17, 026009 (2012). The method has also been applied to the in vitro quantitative evaluation of melanin content on a reconstructed pigmented epidermis (RPE) model of Chinese people, as explained in Qiu, J. et al. “The skin-depigmenting potential of Paeonia lactiflora root extract and paeoniflorin: In vitro evaluation using reconstructed pigmented human epidermis” Int J Cosmet Sci 38, 444 - 451, (2016).

[0010] The intensity-based method for melanin detection disclosed in FR 2944425 is based on the fact that in the skin, at the basal layer level of the epidermis, highly concentrated melanin exhibits a high fluorescence signal intensity stronger than that of other endogenous fluorophores. Pixels with high intensity are attributed to melanin. This intensity-based method works in the basal epidermal layer where melanin is highly concentrated and exhibits a fluorescence signal intensity stronger than that of other endogenous fluorophores, but does not work in the SC containing keratin with high fluorescence intensity. In addition, pixels with low melanin concentration and low fluorescence intensity are not considered.

[0011] A more specific method lies in considering the fluorescence lifetime of melanin. The fluorescence lifetime is independent of the fluorophore concentration but depends on the local microenvironment of the molecule and variables such as pH, binding state, and molecular conformational changes. The autofluorescence lifetime of the skin ranges from hundreds of picoseconds (e.g., melanin, free NAD(P)H, bound FAD) to nanoseconds (e.g., bound NAD(P)H, free FAD, keratin). Multiphoton FLIM imaging of melanin samples such as synthetic melanin, dopa melanin, or sepia melanin, skin and eye melanocytes, human hair and hair bulbs, and human skin has shown specific biexponential decay behavior, with its major (>90% relative contribution) short fluorescence lifetime component being approximately 100 - 200 ps, and the mixture-like phasor plot having a short phase lifetime distribution.

[0012] JP2009 - 142597 describes a method for visualizing melanin by combining multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM). However, the image acquisition time required to obtain the correct fluorescence decay for biexponential analysis or phasor analysis is incompatible with 3D skin imaging in a clinical setting and is limited in practice to 2D imaging at selected epidermal depths, thus restricting the application possibilities of this technique, especially in the case of three-dimensional imaging.

[0013] To specifically detect melanin from 3D multiphoton FLIM-like data, which is also compatible with 3D in vivo acquisition of human subjects, another method is proposed that combines (i) multiphoton FLIM, (ii) fast image acquisition time, and (iii) a melanin detection method called pseudo-FLIM, based on the slope analysis of the autofluorescence intensity decay from time-binned data. WO2013 / 068943 discloses such a method for specifically 3D detecting, visualizing, and / or quantifying endogenous fluorophores such as melanin in biological tissues, which is also described and compared with FLIM and phasor analysis in Pena, A.M. et al. “In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses”. Sci Rep 12, 1642 (2022). Using parameters of melanin 3D total density and z-epidermal distribution, in vivo melanin regulation is evaluated under different conditions: constitutive and acquired pigmentation, aging, natural UV exposure, or the application of topical retinoids known to affect pigmentation.

[0014] However, 3D imaging is very time-consuming and greatly limits its routine use in the assessment of the efficacy of melanin regulators. Multiphoton 2D vertical imaging (acquiring images similar to transverse tissue sections) can be an alternative to a faster melanin evaluation protocol. Although multiphoton XZ imaging has been applied to the characterization of skin aging in human skin (e.g., Czekalla, C. et al. “Impact of Body Site, Age, and Gender on the Collagen / Elastin Index by Noninvasive in vivo Vertical Two-Photon Microscopy”, Skin Pharmacol Physiol 30, 260-267 (2017)), its interest and advantages over 3D imaging in the case of melanin evaluation have not been investigated.

[0015] Overview

[0016] There is still a need to benefit from a sensitive non-invasive method that is easy to implement for the three-dimensional visualization of the distribution of melanin in biological tissues, enabling the obtaining of reliable, fast, and meaningful results.

[0017] The object of the present invention is to develop a method that can be simpler and faster than the prior art methods, which enables the detection of melanin and the characterization of its three-dimensional distribution in biological tissues (especially the epidermis).

[0018] There is also a need to enable the verification of the efficacy or harmlessness of products used during treatment, such as anti-pigmentation agents, depigmentation agents, or pigmentation-promoting agents.

[0019] Furthermore, in the case of seeking to develop new tissues in a reconstructive or synthetic manner, it is necessary to specify the differences in the amount and distribution of melanin according to the type of tissue model (especially skin models).

[0020] The present invention aims to meet all or part of these needs.

[0021] Exemplary embodiments of the present invention relate to a method for detecting, quantifying, and / or visualizing endogenous fluorophores such as melanin in biological tissues, the method comprising:

[0022] - acquiring a plurality of two-dimensional (2D) multiphoton images of fluorescence intensity, or combined multiphoton FLIM images, acquiring each image in a plane substantially perpendicular to the surface of the biological tissue, and

[0023] - processing the images to detect the boundary of the surface of the biological tissue and to detect endogenous fluorophores, especially melanin, and

[0024] - Generate information representing the distribution of endogenous fluorophores in the biological tissue relative to the boundary, in particular as a function of the distance from the boundary, based on the processed image.

[0025] With the aid of the present invention, a new and faster method for melanin evaluation (especially in the RPE model) can be obtained based on the acquisition of a number of transverse 2D XZ images. Each image is, for example, similar or equivalent to the image provided by histological Fontana-Masson staining.

[0026] As is known to those skilled in the art, combined multi-photon FLIM is fluorescence lifetime imaging microscopy (FLIM) using multi-photon excitation. Combined multi-photon FLIM is described, for example, in Periasamy, A. & Clegg, R. M. FLIM Microscopy in Biology and Medicine. 1st Edition (Chapman and Hall / CRC, Taylor & Francis Group, 2009).

[0027] Preferably, fluorescence intensity or pseudo-FLIM analysis is used to detect endogenous fluorophores. As is known to those skilled in the art, fluorescence intensity analysis as described, for example, in FR 2944425 can be used to process multi-photon images for melanin detection. The multi-photon FLIM image processing can be performed for melanin detection and analyzed using any one of FLIM biexponential analysis, phasor analysis, and pseudo-FLIM analysis, and the pseudo-FLIM analysis is described in WO2013 / 068943 and Pena et al. Sci Rep 12, 1642 (2022) above.

[0028] FLIM biexponential analysis is described, for example, in Dancik, Y., Favre, A., Loy, C. J., Zvyagin, A. V. & Roberts, M. S. "Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo" J. Biomed. Opt. 18, 26022; Ehlers, A., Riemann, I., Stark, M. & König, K. "Multiphoton fluorescence lifetime imaging of human hair" Microsc. Res. Tech. 70, 154–161; Dimitrow, E. et al. "Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Exp. Dermatol" 18, 509–515; Sugata, K. et al. "Imaging of melanin distribution using multiphoton autofluorescence decay curves" Skin Res. Technol. 16, 55–59.

[0029] Phasor analysis is described, for example, in Stringari, C. et al., "Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in alive tissue", Proc. Natl. Acad. Sci. U.S.A. 108, 13582–13587; Digman, M.A., Caiolfa, V.R., Zamai, M. & Gratton, E., "The phasor approach to fluorescence lifetime imaging analysis", Biophys. J. 94, L14-16; Vallmitjana, A. et al., "Resolution of 4 components in the same pixel in FLIM images using the phasor approach", Methods and Appl. Fluorescence 8, 035001.

[0030] In the following, the Z direction will indicate the direction along the depth of the biological tissue, substantially perpendicular to the surface. The X and Y directions will indicate two directions defining a plane parallel to the surface of the biological tissue. For example, for the skin, the Z direction is the direction from the basal layer of the skin towards the stratum corneum, or vice versa, and the X and Y directions indicate directions defining a plane parallel to the skin surface. Thus, the XZ image will indicate an image substantially perpendicular to the surface of the biological tissue.

[0031] The melanin density estimated from a subset of 2D XZ images according to the present invention is equivalent to the melanin density estimated in 3D, and the present invention allows for a faster evaluation protocol by acquiring a subset of 2D XZ images instead of a stack of 3D XYZ images.

[0032] Each pixel of the 2D XZ image can be defined by spatial coordinates (x, z), where z is the depth in the sample. For a 2D XZ image, a row of pixels can be acquired at each z position corresponding to a given depth of the biological tissue. The number of pixels and the pixel size (in μm) in the X and Z directions are parameters depending on the imaging system (field of view and resolution). The pixel size in Z depends on the imaging depth and the sample thickness. For example, a 2D XZ image of 1024×1024 pixels can be acquired in the RPE model.

[0033] Preferably, for faster image acquisition times, each XZ image can be acquired with an objective lens that is translatable in the Z direction (e.g., by using a piezoelectric device).

[0034] Preferably, the method of the present invention includes acquiring between 20 and 80, more preferably between 30 and 70, images with different y coordinates within a sample such as an RPE model, e.g., approximately 50 2D XZ images per dy = 100 μm. The number of images and the dy step value (i.e., the acquisition distance between two XZ images along the Y direction) can be adjusted depending on the uniformity of melanin distribution. The method can include acquiring at least fifty two-dimensional (2D) multiphoton images or combined multiphoton FLIM images in different planes substantially perpendicular to the surface of the biological tissue. Preferably, to increase the imaging depth and ensure good fluorescence signal intensity within a biological sample such as an RPE model, its basal layer is placed on the surface of the sample. In current histological Fontana-Masson analysis, only a few 10 - 15 XZ images are acquired and investigated.

[0035] The acquired images can be further processed to detect melanin using methods such as those disclosed in FR 2944425 for intensity-based melanin detection or those disclosed in WO2013 / 068943 for lifetime-based melanin detection. Relevant melanin quantification tools (e.g., computer algorithms) can be utilized to generate further information to extract quantitative parameters of melanin in biological tissue, such as 2D melanin density and / or its z distribution curve.

[0036] The determination of the boundary line between air and the biological tissue surface can be implemented by finding signal points along each column of the XZ image. The method can include detecting the boundary between air and the basal layer, e.g., by determining the median position of the pixels with the highest fluorescence intensity in the surrounding interval in the X direction. The length of the surrounding interval can depend on the image size in the X direction. For a 2D XZ image of 1024×1024 pixels, the length of the surrounding interval along the X direction can be approximately 20 pixels.

[0037] Preferably, the method includes applying a first intensity detection threshold to segment the sample surface for boundary detection. The method can include binarizing the 2D XZ image to denoise the image.

[0038] The boundary line can be displayed on the processed image.

[0039] The method may include, after boundary detection, applying a second intensity detection threshold to binarize and Gaussian blur the 2D image, so as to facilitate detecting melanin-containing pixels using the intensity-based melanin detection method described in FR 2944425. In the living epidermis, melanin pixels with strong fluorescence intensity showing a fluorescence intensity level higher than that of other endogenous fluorophores (such as NAD(P)H in the stratum granulosum) are selected by applying an intensity threshold. Preferably, the second intensity detection threshold is set to a value higher than the first boundary detection threshold. Gaussian blur is applied to allow elimination of noise pixels, and then the second intensity threshold is applied for melanin detection. The second intensity threshold may be the same as that taught in 3D imaging (such as in the above-mentioned FR 2944425 patent and the article “The skin-depigmenting potential of Paeonia lactiflora root extract and paeoniflorin: In vitro evaluation using reconstructed pigmented human epidermis”). The average number of melanin-containing pixels relative to their distance from the basal epidermal boundary can be used to estimate the melanin z-distribution curve from the basal layer up to a depth of, for example, 100 μm.

[0040] The association of the 2D XZ acquisition with the melanin quantification tool according to the present invention allows visualization and quantification of the distribution and amount of melanin in the RPE epidermal model, which is, for example, at least 5 times faster in operation time compared to 3D imaging, thus providing a powerful method for the routine efficacy evaluation of melanin regulators in cosmetic and dermatological research.

[0041] The method is non-invasive and can be used in non-therapeutic, cosmetic situations.

[0042] The method enables the possible evaluation of pigmentation.

[0043] The method according to the present invention can be implemented in vivo and can also be implemented on ex vivo samples and in vitro samples.

[0044] Compared with 3D imaging, the present invention improves the speed of the melanin efficacy evaluation process by reducing the number of XZ images required for robust estimation of melanin density in the RPE model. The method also allows improvement in the speed of processing images for generating information representing the distribution of endogenous fluorophore components in the biological tissue. With the present invention, 3D characterization of tissues is possible and can be used for in vivo applications at high throughput.

[0045] The biological tissue (especially keratin material) according to the present invention can be natural or artificial; the sample is, for example, human skin, or reconstructed skin or artificial skin.

[0046] The biological tissue can be cultured melanized cells.

[0047] Information can be generated in the form of at least one image, in particular a two-dimensional or three-dimensional image.

[0048] Compared with other tissue components, the generated information can provide information about the surface and / or volume occupied by melanin in the tissue, as well as information about the 3D distribution of melanin.

[0049] Exemplary embodiments of the present invention also relate to a method (in particular a non-therapeutic method) for evaluating the effect of a stimulus and / or treatment that promotes pigmentation, depigmentation or anti-pigmentation on biological tissue, which includes:

[0050] - Evaluating pigmentation in biological tissue by means of the method described above, subjecting the sample to a stimulus (in particular a non-therapeutic stimulus) selected from: light radiation (in particular solar radiation, ultraviolet (UVA and / or UVB) radiation or infrared (IR) radiation), a stimulus that causes an inflammatory reaction, and mechanical actions (stretching, pressing, peeling, separating, flaking, abrasion), or subjecting the sample to a treatment, in particular a treatment with at least one product that promotes pigmentation, depigmentation or anti-pigmentation (such as lucinol or any other chemical reagent that regulates pigmentation).

[0051] - Performing a second evaluation of pigmentation in biological tissue by means of the method described above.

[0052] - Comparing the information generated during the two evaluations and evaluating the effect of the stimulus or treatment on pigmentation based at least on this comparison.

[0053] The treatment can be non-therapeutic, in particular cosmetic.

[0054] The treatment can be selected from: application, injection, ingestion or inhalation of a product (in particular a cosmetic product).

[0055] The treatment can correspond to taking a food supplement and / or a medicament. The treatment can also include subjecting the biological tissue to a treatment selected from: application, injection, ingestion or inhalation of a product (in particular a cosmetic product), or taking a food supplement and / or a medicament.

[0056] The term "cosmetic product" is intended to mean a product as defined in Directive 93 / 35 / EEC of 14 June 1993 (which amends Directive 76 / 768 / EEC).

[0057] The product can have a depigmenting effect. Thus, the treatment can include the application of any chemical reagent that regulates pigmentation.

[0058] The method according to the invention can be used to evaluate the efficacy or harmlessness of depigmenting agents, anti - pigmentation agents or pigmentation - promoting agents. The agents can be used for therapeutic purposes (such as hydroquinone or retinoids) and / or cosmetic purposes.

[0059] The method according to the invention can also be used to evaluate the side effects on pigmentation of certain products (such as dermocorticoids).

[0060] Exemplary embodiments of the invention also relate to a method for evaluating the action of stimuli and / or treatments, in particular anti - pigmentation, pigmentation - promoting or depigmenting, on biological tissue, wherein at least two regions of the tissue are differently subjected to the stimuli and / or are differently treated, and wherein information on the presence and / or quantity of melanin in said regions, obtained by the method as described above, before and after being subjected to said stimuli or said treatment, is compared.

[0061] To test the action of a product, the evaluation made after treatment with a placebo of the product can be compared with the evaluation made after treatment with such a product. The placebo is, for example, a cosmetic medium identical to the cosmetic medium of the product used for treatment, but without the corresponding active agent.

[0062] It is possible to evaluate the distribution of melanin in the biological tissue of an individual who has been treated with a cosmetic compound or on a sample of artificial skin or reconstituted skin, and compare it with the evaluation of the distribution of melanin in the biological tissue of the same individual who has been treated with a placebo of the cosmetic compound or on the same sample of artificial skin or reconstituted skin.

[0063] The treatment can correspond to the application of a product, in particular a cosmetic product. The product can be, for example, in the form of a cream, a lotion, an ointment, an oil, a powder, and the list is not restrictive.

[0064] The product can also be incorporated in a support to be applied to biological tissue (in particular reconstituted human skin or artificial human skin), the support being, for example, a patch, a dressing, a bandage or a mask.

[0065] The product can be not only a pigmentation product or a depigmentation product intended to act on the quantity and / or distribution of melanin in biological tissue. Thus, the product can be, for example, intended for cosmetic use, for moisturizing or for protecting biological tissue (in particular against sunlight), or for repairing biological tissue, while having an effect on the quantity and / or distribution of melanin in biological tissue. Thus, the product can contain various compounds, in particular active agents other than the active agents intended to act on the melanin of biological tissue.

[0066] Exemplary embodiments of the present invention also relate to a method for promoting treatment (especially non-therapeutic treatment), wherein mention is made of the effect of such treatment on melanin as demonstrated by the method defined above. Brief Description of the Drawings

[0068] The present invention can be better understood from reading the following description of non-limiting examples of embodiments of the present invention and viewing the diagrams of the accompanying drawings, wherein:

[0069] - Figure 1 Illustrates the various steps of the method according to the present invention,

[0070] - Figure 2 Illustratively and partially represents an example of a multiphoton device that can be used in the method according to the present invention,

[0071] - Figure 3 Illustrates an example of a multiphoton 2D XZ fluorescence intensity image obtained according to the present invention,

[0072] - Figure 4 Illustrates the Figure 3 image to which boundary detection is applied,

[0073] - Figure 5 Corresponds to the Figure 4 image obtained after melanin detection based on fluorescence intensity,

[0074] - Figure 6 Illustrates the Figure 5 image after aligning the boundaries in the Z direction,

[0075] - Figure 7 Shows the melanin 2D z-distribution curve corresponding to the Figure 6 image in.

[0076] Detailed Description

[0077] As Figure 1 illustrated, step 1 of the method according to the present invention includes acquiring a plurality of two-dimensional XZ images by multiphoton microscopy or multiphoton FLIM microscopy. Each image contains multiple rows of pixels imaged at different depths of biological tissue.

[0078] The use of a multiphoton microscopy device allows for the automation of the biological tissue image acquisition step.

[0079] As mentioned above, the number of 2D XZ images to be acquired and their dy step size are parameters to be adjusted depending on the biological sample and the imaging system. For the RPE skin model, a stack of 50 images can be acquired for every dy = 100 μm. The acquired images can be stored in different formats, such as as individual images or as image stacks. Different sample X, Y positions can also be used to acquire two or more stacks of 2D XZ images.

[0080] In step 2, an algorithm is used to process the image stack and read a single layer from the image stack. Figure 3 Examples of 2D XZ images obtained for the skin by the method of the present invention are illustrated in. The image shows the epidermal fluorescence intensity of the RPE skin model in a transverse plane substantially perpendicular to the skin surface. In the current example, the XZ image to be processed contains a total number of 1024×1024 pixels.

[0081] Step 3 includes boundary determination or detection of biological tissue. This step includes applying a first intensity detection threshold to binarize the extracted 2D XZ image, i.e., denoising it for boundary detection.

[0082] After denoising, the algorithm finds the signal points along the x-axis in each column where the pixels are located and determines the median position of the pixels with the highest fluorescence intensity in the surrounding interval. Then, the algorithm can connect the signal points corresponding to the pixels with the highest fluorescence intensity. The connected signal points form a boundary line, i.e., the boundary between air and the basal layer. The boundary determined in this way is illustrated in Figure 4 in.

[0083] Step 4 includes detecting melanin in the boundary-detected 2D XZ image. This step includes setting a second intensity detection threshold, which is also used for binarization to denoise. Then, Gaussian blur is applied to assist in noise reduction to help detect melanin signal points. As shown in Figure 5 the bright parts are the melanin signal points detected after noise reduction. Therefore, after applying the second intensity detection threshold using a fluorescence intensity-based melanin detection method, a melanin mask is obtained.

[0084] In step 5, a curve representing the melanin distribution in the transverse plane corresponding to the single layer of the 2D XZ image extracted in step 2 is calculated. In the current example, the algorithm takes out 256 pixels from the starting point on the boundary line in each column of pixels and aligns the starting points to form the picture shown in Figure 6 in. Thus, the algorithm detects the skin area. The alignment of the starting points can facilitate the generation of a curve representing the melanin distribution (as illustrated in Figure 7 ). The number of pixels taken out can vary, for example, as a function of the melanin signal points detected in step 4.

[0085] Then, these melanin pixels can be counted based on their distances from the boundary line. The above steps can be applied to at least a part or every one of the acquired 2D XZ images. Then, for each depth corresponding to the distance of a row of pixels taken out in step 5 from the boundary line, by averaging the amount along the X direction in each 2D XZ image and this quantity among the 2D XZ maps, the melanin distribution from the basal layer of the skin to the deep layer can be obtained, as Figure 7 shown, where the abscissa represents the distance from the basal layer to the skin surface (in microns), and the ordinate represents the average value of the melanin pixel counts in the 2D XZ image at a given depth z. Different XZ images can be acquired at different Y positions to facilitate giving information about the melanin distribution at different sample positions. In this example, the melanin 2D z distribution curve is presented from the sample surface to a depth of 250 μm.

[0086] The above method can be implemented using any known multiphoton microscopy system that is combined or not combined with a system for measuring fluorescence lifetime. In particular, the multiphoton microscopy system is, for example, type A1RMP / FN1.

[0087] The excitation wavelength used can be between 700 nm and 1000 nm, preferably about 760 nm. This wavelength range makes it possible to image most of the endogenous fluorescence components of the tissue.

[0088] Figure 2 Illustratively and locally represents an example of a multiphoton device 100 that can be used in the method according to the present invention.

[0089] The device 100 includes a femtosecond laser 10, such as a titanium-sapphire (Ti:Sa) laser, which can be tuned to the infrared wavelength range and can provide pulses of about 100 femtoseconds at a repetition rate of about 80 MHz. The laser 10 emits an infrared beam 11, which is directed towards a laser beam scanning device 12 in the form of an "XY scanner".

[0090] Then, the beam is reflected by a first dichroic mirror 14 and focused on the keratin material 13 by means of an objective lens 15. Scanning of the laser beam can be obtained by the angular movement of the first pair of two galvanometer mirrors 22 of the scanning device 12, which allows scanning of the focal point in a plane (x, y) perpendicular to the Z direction. The piezoelectric device 26 makes it possible to translate the objective lens 15 during the acquisition of the 2D XZ image and thus change the focal plane at different depths in the keratin material 13. For each depth, a row of pixels is acquired by moving the focal point in the X direction by the XY scanner.

[0091] Then, the signal generated at the focus can be detected, for example, via epicollection through the excitation objective 15. The first dichroic mirror 14 enables the selection of the generated multi-photon signal, in particular the autofluorescence (AF) originating from the keratin material 13. In particular, the first dichroic mirror 14 allows the reflection of the laser wavelength (for example, in the range of 700 - 1000 nm) and the transmission of the multi-photon signal (for example, 350 - 650 nm).

[0092] Then, the second dichroic mirror 16 enables the separation of the autofluorescence (AF) signal from other multi-photon signals, for example, corresponding to second harmonic generation (SHG).

[0093] A second pair of two galvanometric mirrors 23 can be located between the first dichroic mirror 14 and the second dichroic mirror 16 for performing optical alignment to ensure that all signals can be detected by the detector regardless of the XZ position.

[0094] In any case, the signal passes through the spectral filter and reaches at least one detector 18, thus enabling the generation of a fluorescence image. The data is further transmitted to another computer for image processing.

[0095] Therefore, this method can be used for any type of tissue containing melanin.

Claims

1. A method for detecting, quantifying, and / or visualizing endogenous fluorophores such as melanin in biological tissue, the method comprising: - obtaining a plurality of two-dimensional (2D) multiphoton images of fluorescence intensity, or combined multiphoton FLIM images, each image being obtained in a plane substantially perpendicular to the surface of the biological tissue, - processing the images to detect the boundary of the surface of the biological tissue and to detect the endogenous fluorophore, and - generating, based on the processed images, information representing the distribution of the endogenous fluorophore in the biological tissue relative to the boundary, in particular as a function of the distance from the boundary.

2. The method according to claim 1, the method comprising obtaining at least fifty two-dimensional (2D) multiphoton images or combined multiphoton FLIM images in different planes substantially perpendicular to the surface of the biological tissue, preferably using fluorescence intensity or pseudo-FLIM analysis to detect the endogenous fluorophore.

3. The method according to claim 1 or 2, generating the information in the form of at least one representation of the 2D melanin density and / or its distribution curve along the depth of the biological tissue.

4. The method according to any one of the preceding claims, which comprises applying a first intensity detection threshold to segment the sample surface for boundary detection.

5. The method according to any one of the preceding claims, which comprises determining the boundary line between air and the surface of the biological tissue by finding signal points along the columns of the 2D image and determining the median position of the pixels with the highest fluorescence intensity in the surrounding interval.

6. The method according to any one of the preceding claims, which comprises displaying the boundary line on the processed image.

7. The method according to any one of the preceding claims, which comprises, after boundary detection, applying a second intensity detection threshold to binarize and Gaussian blur the 2D image in order to facilitate the detection of pixels containing melanin.

8. The method according to the preceding claim, which comprises taking pixels from the starting point on the boundary line in each column of pixels of the 2D image and aligning the starting points.

9. The method according to any one of the preceding claims, wherein the biological tissue is skin and the endogenous fluorophore component is melanin, the method comprising representing the melanin distribution as a function of the distance from the basal layer of the skin.

10. The method according to any one of the preceding claims, which comprises obtaining multiple rows of pixels at different depths of the biological tissue for each 2D image, the method comprising, each time a row of pixels is obtained, translating an objective lens perpendicular to the surface of the biological tissue during the acquisition of each 2D image, the objective lens being focused on the corresponding depth of the biological tissue.

11. The method according to any one of the preceding claims, the generated information providing information about the surface and / or volume occupied by melanin in the tissue.

12. The method according to any one of the preceding claims, which is implemented in vivo on biological tissue.

13. The method according to any one of the preceding claims, wherein the tissue is human skin, reconstructed skin or artificial skin, or cultured melanized cells.

14. A method for evaluating the effect of a stimulus and / or treatment for promoting pigmentation or depigmentation, in particular, on biological tissue, comprising: evaluating pigmentation in the tissue by implementing the method according to any one of the preceding claims; performing a second evaluation of the pigmentation in the tissue by the method according to any one of the preceding claims, wherein the tissue has been subjected to the action of a stimulus selected from the following: light radiation, in particular solar radiation, ultraviolet (UV) radiation or infrared (IR) radiation; a stimulus causing an inflammatory reaction, and mechanical action; or the tissue has been subjected to a treatment, in particular has been treated with at least one pigmentation-promoting or depigmenting cosmetic, comparing the information generated during the two evaluations, and evaluating the effect of the stimulus or the treatment on pigmentation at least based on this comparison.

15. A method for evaluating the effect of a stimulus and / or treatment for anti-pigmentation, promoting pigmentation or depigmentation, in particular, on biological tissue, wherein at least two regions of the tissue are differently subjected to the stimulus and / or are differently treated, and wherein information on the presence and / or amount of melanin in the regions, obtained by means of the method according to any one of claims 1-10, is compared before and after the tissue has been subjected to the stimulus or the treatment.

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