WO2013068943A1

WO2013068943A1 - Non-invasive method for specific 3d detection, visualization and/or quantification of an endogenous fluorophore such as melanin in a biological tissue

Info

Publication number: WO2013068943A1
Application number: PCT/IB2012/056230
Authority: WO
Inventors: Thérèse Baldeweck; Anna-Maria PENA; Emmanuelle TANCREDE-BOHIN; Etienne Decenciere; Serge KOUDORO
Original assignee: L'oreal
Priority date: 2011-11-08
Filing date: 2012-11-07
Publication date: 2013-05-16
Also published as: CN103930767B; KR102024956B1; CN103930767A; JP6147756B2; FR2982369B1; JP2014532888A; KR20140090184A; FR2982369A1

Abstract

Method for detection, quantification and/or visualization of an endogenous fluorophore, in a biological tissue, the method comprising the steps consisting in: a) acquiring, after multiphoton excitation, a set of successive three- dimensional or two-dimensional images providing information on the decrease over time in the fluorescence signals in the sample, b) determining by processing these images, by performing a linear regression of the logarithm of the fluorescence signals, the spatial distribution in the sample of the slopes of said linear regression, and c) generating information on the presence and/or the volume density or surface density of a fluorophore in the sample at least on the basis of this spatial distribution, the fluorophore being melanin.

Description

NON-INVASIVE METHOD FOR SPECIFIC 3D DETECTION, VISUALIZATION AND/OR QUANTIFICATION OF AN ENDOGENOUS FLUOROPHORE SUCH AS MELANIN IN A BIOLOGICAL TISSUE

The present invention relates to the observation of biological tissues, in particular keratin materials such as the skin.

The invention relates more particularly but not exclusively to the methods aimed at determining the distribution, in particular the amount and/or the nature of distribution, of melanin in biological tissues, with a view in particular to evaluating the action of a cosmetic or therapeutic treatment.

Background

The colour of biological tissues is closely linked to the amount and the three-dimensional distribution of melanin. Today, the characterization of the amount and of the distribution of melanin can be carried out either in two-dimensional form by white light imaging and quantification of the melanin stained with Fontana Masson on a histological section, or in three-dimensional form by multiphoton imaging and three-dimensional quantification of the melanin after image processing.

Multiphoton microscopy facilitates the deep observation of biological tissues, in particular highly scattering live tissues. This is because infrared light penetrates better into tissues since it is scattered less and absorbed less, and using a non-linear mechanism to produce the signal introduces a stronger signal selection criterion in a scattering medium. This non-linear mechanism involves the simultaneous interaction of two or even several photons with the molecule or the structure that it is desired to detect. Generally, the excitatory beam is focused and scanned in the sample, and the non-linear signal created is detected in order to obtain an image. The particularity of this type of imaging is based on the optical section capacity that it provides owing to the non-linear dependence of the signal with the excitation power. Since the excitation power density is very high at the focal point, the non-linear effect under consideration occurs efficiently only in a limited focal volume; this essential property ensures containment of the signal obtained in the sample and therefore makes it possible to obtain an intrinsic three-dimensional resolution of the order of a micrometre. The imaging depth varies according to tissues, it may be approximately 130-200 μιη for human skin. In addition, because of the use of endogenous signal sources, this imaging technique does not require labelling of the tissue components. The fluorescence signals are created by endogenous chromophores; in addition to melanin, mention may be made of NAD(P)H, flavins, keratin and elastin.

Application FR 2 944 425 discloses a method for evaluating skin pigmentation by multiphoton microscopy based on the fact that, in the skin, at the level of the basal layers of the epidermis, the highly concentrated melanin is reflected by a fluorescence intensity that is stronger than that of the other endogenous fluorophores. The pixels which have a strong intensity are attributed to melanin. Nevertheless, this method is not always satisfactory, since, firstly, it can be disrupted by other fluorophores which also exhibit a strong fluorescence intensity, such as keratin, and, secondly, it does not take into account melanin exhibiting a fluorescence signal comparable to that of the other endogenous fluorophores.

A more specific method consists in taking into account the fluorescence lifetime of melanin. Indeed, the fluorescence lifetime of melanin depends on the intrinsic fluorescence properties of melanin and not on the intensity of fluorescence nor on the concentration of the fluorophore in the excitation volume. Melanin has a two-photon excited fluorescence lifetime which is different from that of the other endogenous fluorophores. This fluorescence lifetime may be estimated on the basis of images acquired by FLIM (Fluorescence Lifetime Imaging), a technique described in the article by M.S. Roberts et al, "Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy" (European Journal of Pharmaceutics and Biopharmaceutics vol. 77 (2011), pp 469- 488), which consists in monitoring the decrease in the fluorescence signal over time. Application JP2009-142597 describes a method for visualizing melanin which combines multiphoton microscopy and FLIM. The latter technique, in order to provide a good estimation of the fluorescence lifetime, requires integration of the fluorescence signal in time and space, a costly operation in terms of time, thereby limiting the possibilities of application of this technique, in particular in the context of three- dimensional imaging. Summary

There is thus a need to have the benefit of a sensitive, non-invasive method which is easy to implement, for three-dimensional visualization of the distribution of melanin in biological tissues, making it possible to obtain reliable, fast and relevant results.

The objective of the present invention is to develop a method which is simpler and faster than the prior art methods, which makes it possible to specifically detect melanin and to characterize its three-dimensional distribution in a biological tissue, in particular the epidermis.

There is also a need to make it possible to verify the efficacy or the innocuousness of a product, for example based on anti-pigmenting, depigmenting or pro- pigmenting active agents, used during treatment.

There is also an advantage in characterizing pigmentation, in particular of the skin, in order to objectify the differences in amount and distribution of melanin according to skin type, population, geographical location, or else diet, etc., and in order to characterize pigmenting disorders, in particular of the skin, such as actinic lentigenes, ephelides (freckles) or melasmas (mask which sometimes appears during pregnancy).

In addition, in the context of the search to develop new tissues in a reconstructed on synthetic manner, there is a need to objectify the differences in amount and distribution of melanin, according to the type of tissue model, in particular of skin model.

The invention aims to meet all or part of these needs.

The subject of the invention, according to one of its aspects, is thus a method for detection, quantification and/or visualization of an endogenous fluorophore component, such as melanin, in a biological tissue, the method comprising the steps consisting in:

a) acquiring, after multiphoton excitation, a set of successive three- dimensional or two-dimensional images providing information on the decrease over time in the fluorescence signals in the sample,

b) determining by processing these images, by performing a linear regression of the logarithm of the fluorescence signals, the spatial distribution in the sample of the slopes of said linear regression, and c) generating information on the presence and/or the amount of the fluorophore in the sample at least on the basis of this three-dimensional spatial distribution.

The method is non-invasive and may be used in a non-therapeutic, cosmetic context.

The method may make it possible to evaluate pigmentation.

The images acquired are three dimensional temporal images. The term "three- dimensional temporal image" is intended to mean a series of successive three-dimensional images over time.

Each three-dimensional image corresponds to a stack of two-dimensional images representative, at a given moment and a given depth, of the structure of the sample.

Each pixel of each three-dimensional or two-dimensional image may be defined by spatial coordinates (x, y, z), z being the depth in the sample. Thus, each two- dimensional image corresponds to a particular case of a three-dimensional image, in which the acquisition is carried out solely at a given depth z.

The pixels of the three-dimensional images are grouped together in sets of pixels having the same spatial coordinates of images taken at various moments, each of these sets being called a "temporal pixel".

The value of the fiuorescence signal of each pixel may be obtained by integration over a time period of the two-photon excited fluorescence signal. The integration of the fluorescence signal can in particular be carried out at regular intervals, in particular over a period of between 1 and 3 ns, for example equal to 1, 1.5, 2, 2.5 or 3 ns.

The slope of decrease in fluorescence signals can be obtained by linear regression of the logarithm of the fiuorescence lifetimes. The method may require the acquisition only of a limited number of three-dimensional temporal images, for example between three and five, which offers a good compromise between acquisition time and precision. The use of the slopes obtained by linear regression of the logarithm facilitates this limitation of the number of acquisitions.

Compared with the FLIM technology, the invention reduces the number of temporal acquisitions and makes the method easier and faster to implement. While the FLIM technology allows a better estimation of the fluorescence lifetime, but requires quite a long acquisition time (approximately 30 s) with degradation of the resolution of the three-dimensional image, the method according to the invention is faster and particularly advantageous for obtaining three-dimensional visualization or quantification, while at the same time keeping a resolution equivalent to the theoretical resolution of the microscope.

The total acquisition time for the temporal pixels of coordinates (x, y, z) of a given sample is approximately 0.28 instead of a few ms in FLIM.

The number of temporal acquisitions for each temporal pixel is, for example, equal to four, and for each pixel, the two-photon excited fluorescence signal is integrated over time between two acquisitions, in particular with a regular interval of 2 ns.

The method according to the invention can be implemented in vivo, but also on ex vivo and in vitro samples.

The invention improves the rapidity of the acquisition method and of the image processing method. By virtue of the invention, 3D characterization of the tissue is possible and allows an in vivo application with a high throughput.

The tissue may consist of human keratin materials. The term "keratin materials" is intended to mean the hair, the eyelashes, the eyebrows, the skin, the nails, the mucous membranes, the scalp, inter alia.

The biological tissue, in particular the keratin materials, according to the invention may be natural or artificial; the sample is, for example, human skin, or reconstructed or artificial skin.

The biological tissue may be melanized cells in culture.

The information may be generated in the form of at least one image, in particular a two-dimensional or three-dimensional image.

The information generated may provide information on the surface and/or the volume taken up by melanin in said tissue, and also on its 3D distribution compared with the other constituents of the tissue.

A subject of the invention, according to another of its aspects, is a method for evaluating the action of a stimulus and/or of a treatment, which is in particular pro- pigmenting, depigmenting or anti-pigmenting, on a biological tissue, comprising the steps consisting in:

evaluating the pigmentation in a biological tissue by means of a method as previously described, exposing the sample to a stimulus chosen from: light radiation, in particular solar, ultraviolet (UVA and/or UVB) or infrared (IR) radiation, a stimulus causing an inflammatory response, and a mechanical action (tension, pressure, peeling, detachment, exfoliation, abrasion), or to a treatment, in particular with at least one pro- pigmenting, depigmenting or anti-pigmenting product,

performing a second evaluation of the pigmentation in the biological tissue by means of a method as previously described,

comparing the information generated during the two evaluations, and evaluating the action of the stimulus or of the treatment on the pigmentation on the basis at least of this comparison.

The treatment may be non-therapeutic, in particular cosmetic.

The treatment may be chosen from: the application, injection, ingestion or inhalation of a product, in particular a cosmetic product.

The treatment may correspond to taking food supplements and/or medicaments. It may also comprise exposure of the biological tissues to a treatment chosen from: the application, injection, ingestion or inhalation of a product, in particular of a cosmetic product, or the taking of food supplements and/or of medicaments.

The term "cosmetic product" is intended to mean a product as defined in Directive 93/35/EEC of 14 June 1993, modifying Directive 76/768/EEC.

The product may have a depigmenting effect. Thus, the treatment may comprise the application of any pigmentation-modulating chemical agent.

The method according to the invention may be used for evaluating the efficacy or the innocuousness of depigmenting, anti-pigmenting or pro -pigmenting active agents. The active agents may be for therapeutic purposes, for instance hydroquinone or retinoids, and/or cosmetic purposes.

The method according to the invention may also be used for evaluating the side effects on pigmentation of certain products, for example dermocorticoids.

A subject of the invention, according to another of its aspects, is a method for evaluating the action of a stimulus and/or of a treatment, which is in particular anti- pigmenting, pro-pigmenting or depigmenting, on a biological tissue, in which at least two regions of the tissue are exposed differently to the stimulus and/or treated differently, and in which the information on the presence and/or the amount of melanin in said regions, obtained by means of a method as previously described, before and after exposure to said stimulus or to said treatment, are compared.

To test the action of a product, an evaluation made after treatment with a placebo of the product and an evaluation made after treatment with this product may be compared.

The placebo is, for example, the same cosmetic medium as that of the product used for the treatment, but without the corresponding active agent(s).

It is possible to carry out an evaluation of the distribution of melanin in the biological tissues of an individual or on samples of artificial or reconstructed skin which have been treated with a cosmetic compound, and to compare with an evaluation of the distribution of melanin in the biological tissues of the same individual or the same samples of artificial or reconstructed skin which have been treated with a placebo of the cosmetic compound.

The treatment may correspond to the application of a product, in particular a cosmetic product. The product may, for example, be in the form of a cream, a lotion, an ointment, an oil, a powder, this list not being limiting.

The product may also be contained in a support to be applied to the biological tissues, in particular reconstructed or artificial human skin, for example a patch, a dressing, a bandage or a mask.

The product may not be a pigmenting or depigmenting product solely intended to act on the amount and/or the distribution of melanin in the biological tissues. The product may thus be intended, for example, for making up, for moisturizing or for protecting biological tissues, in particular protecting against the sun, or for repairing biological tissues, while at the same time having effects on the amount and/or the distribution of melanin in the biological tissues. The product may thus contain various compounds, in particular active agents other than active agents intended to act on the melanin of biological tissues.

The method may make it possible to visualize melanin in the biological tissues; it may, for example, make it possible to know the distribution of melanin in the various layers of the epidermis of the skin.

Another subject of the invention, according to another of its aspects, is a method for promoting a treatment, in particular a non-therapeutic treatment, in which reference is made to an action of the treatment on melanin, demonstrated by means of a method as defined above.

Another subject of the invention, according to another of its aspects, is a method comprising the step consisting in making reference, during the marketing of a product, for example in the advertising or on packaging, to the fact that the efficacy of the product was verified by means of a method as defined above.

The invention may be better understood from reading the following description of non-limiting examples of implementation thereof, and from examining the figures of the attached drawing, in which:

- Figure 1 illustrates various steps of a method in accordance with the invention,

Figure 2 represents, diagrammatically and partially, an example of a multiphoton device which may be used in a method according to the invention,

Figures 3A to 3D illustrate an example of image acquisition according to the invention,

Figures 4 A and 4B illustrate various examples of temporal change in fluorescence inside a sample,

Figure 5 illustrates a parametric representation of the fluorescence lifetimes for a section of a skin sample at a fixed depth,

- Figures 6 and 7 illustrate two-dimensional and three-dimensional masks of melanin distribution in a skin sample, obtained by means of a method according to the invention,

Figure 8 illustrates a two-dimensional mask of melanin distribution in a skin sample, obtained by means of a method of the prior art,

- Figures 9A to 9D illustrate the amount of melanin in a skin sample before and after a treatment, and

Figures 10A, 10B and IOC illustrate the characterization of human skin phototypes I to IV by the amount of melanin.

Step 1 of a method according to the invention may consist in acquiring, by multiphoton microscopy, sets of two-dimensional temporal images at various depths of a biological tissue sample. Each pixel may be obtained by integration over a period of time of the two- photon excited fluorescence signal, for example using a TCSPC (time-correlated single photon counting) photon counting device.

The use of a multiphoton microscopy device may allow automation of the biological tissue image acquisition steps.

A three-dimensional temporal image may in particular be generated from a stack of two-dimensional temporal images at various depths. Each stack may comprise several tens of two-dimensional temporal images, for example between 50 and 100 images, in particular between 65 and 80 images.

In a step 2, for each pixel of coordinates (x, y, z), the variation in fluorescence intensity is studied as a function of time by calculating its logarithm. The logarithm is subsequently adjusted by means of a linear regression in order to estimate the slope thereof, which is related to the fluorescence lifetime.

The data obtained may thus be represented by an image of the slopes obtained by linear regression of the logarithm of the fluorescence intensity.

The slope image, also called parametric image, contains two types of pixels: pixels characterized by a low slope, and pixels characterized by a high slope value. The pixels having high-value slopes are attributed to melanin, which has a shorter lifetime compared with the other endogenous fluorophores of the cells of the epidermis, such as keratin, NAD(P)H, FAD, etc.

Indeed, melanin is characterized by two fluorescence lifetimes. The main one, which is very short, is of the order of 0.1-0.2 ns (nanosecond). The other fluorescence lifetime of melanin is minor and longer, between 0.7 and 1.4 ns. By comparison, the other endogenous fluorophores of the cells of the epidermis, such as keratin, NAD(P)H, FAD, etc., have predominantly longer fluorescence lifetimes, greater than 1.5 ns.

Step 3 consists in extracting, from the slope image, the pixels corresponding to melanin. Surface filtering is, for example, applied to each two-dimensional image, which takes into account the size of the melanosomes, i.e compares it with a reference surface area of approximately 1 μιη², in order to remove the noise and to more specifically detect the melanin. The information on the presence and/or the amount of melanin in the sample is, for example, generated after having eliminated the fluorescence signals having low- value slopes and the structures having a size less than 1 μιη².

The information may be generated in the form of at least one representation of the spatial distribution of melanin, in particular in the form of a two-dimensional image corresponding to the melanin mask for one depth of the sample.

Step 2 of calculating the slopes and step 3 of filtering the associated mask may be carried out on the whole of the two-dimensional image corresponding to a given depth, and repeated for each image of the three-dimensional stack.

The method makes it possible to obtain a stack of two-dimensional representations forming a three-dimensional image of the melanin mask.

On the basis of the three-dimensional melanin mask obtained by means of this method, it is possible to characterize the melanin by calculating its density and/or its spatial distribution.

Given the rapidity of the calculations, this method may be used in a screening method.

Any known multiphoton microscopy system, combined with a system for measuring fluorescence lifetime, may be used to implement the method above.

The excitation wavelength used may be between 700 and 1000 nm, preferably about 760 nm. This wavelength range makes it possible to image most of the fluorescent constituents of tissues.

Figure 2 represents, diagrammatically and partially, an example of a multiphoton device 100 which may be used in a method according to the invention.

The multiphoton device 100 is, for example, of the Dermalnspect^® type developed by the company Jenlab.

The device 100 comprises a femtosecond laser 10, for example a Titanium- Sapphire (Ti : Sa) laser, which may be tuned into an infrared wavelength range and which may provide pulses of the order of 100 femtoseconds at a repetition rate of the order of 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". A scan of the laser beam may be obtained by angular movement of two galvanometric mirrors of the scanning device 12. The beam is then reflected by a dichroic mirror 14 and it is focused on the keratin materials 13 by means of the objective 15. Thus, the two galvanometric mirrors allow scanning of the focusing point in the plane (x, y) perpendicular to the direction of propagation of the beam (z axis).

A piezoelectric device makes it possible to translate the objective 15 and thus to change the plane of focusing in the keratin materials 13. In this way, it is possible to reconstitute the three-dimensional distribution of melanin in the keratin materials by superposition of the images acquired.

To do this, the signals created at the focal point may then be detected, that is to say by epicollection through the excitation objective 15. The first dichroic mirror 14 makes it possible to select the multiphoton signals created, in particular the auto fluorescence (AF) originating from the keratin materials 13.

Then, a second dichroic mirror 16 makes it possible to separate the autofluorescence (AF) signals from the other multiphoton signals, corresponding for example to the second harmonic generation (SHG). In any event, the signals pass through spectral filters 17 and reach the photon counting detectors (TCSPC: time-correlated single photon counting) 18, 19, thus making it possible to produce a combined image, for example an AF/SHG image.

The first and second detectors 18 and 19 make it possible to generate signals 18a and 19a which are transmitted to an electronic device 20 for signal acquisition and processing.

Example 1 : in vivo observation of human skin

During tests, three-dimensional images were successively obtained in vivo in humans on the forearm, one three-dimensional image consisting of a stack comprising 70 two-dimensional images acquired at depths ranging from 2.346 μιη from the surface of the skin, obtained using the Dermalnspect^® device, with an excitation wavelength of 760 nm, and a 40x/1.3 NA objective.

Figures 3 A to 3D illustrate a temporal sequence of three-dimensional images, of which are shown only four two-dimensional temporal images acquired in vivo on the skin of the forearm of a healthy volunteer, close to the basal layer of the epidermis at a depth of 50 μιη relative to the surface of the skin. For each temporal image, the fluorescence signal was integrated over a period of 2 ns.

Under the experimental conditions, for a point of the sample containing melanin, the majority of the fluorescence photons emitted are collected during the first temporal acquisition. The greater the concentration of melanin, the higher the number of photons of this first acquisition. The arrow I of Figure 3A, corresponding to the first temporal channel, indicates an area of high melanin concentration, on the basis of the intensity criterion. At this level, the highly concentrated melanin creates a fluorescence signal of stronger intensity than that of the other endogenous fluorophores.

Figures 4A and 4B show an example of a temporal evolution curve (Figure 4 A) and of adjustment of the logarithm of fluorescence intensity by means of a linear regression (Figure 4B) for a pixel of the image corresponding to a point of the sample with melanin and for a pixel corresponding to a point of the sample without melanin.

Figures 5, 6 and 8 correspond to two-dimensional representations of human skin at a depth of 50 μιη. Figure 5 shows a parametric image of the slopes obtained by linear regression of the logarithm of the fluorescence intensity. The image has two types of pixels: pixels characterized by a low slope, and pixels characterized by a high slope value, attributed to melanin for the reasons set out previously.

Figure 6 shows the two-dimensional mask of melanin obtained from Figure 5 by extraction of the pixels attributed to melanin, while Figure 8 illustrates the result obtained with the prior art method solely on the basis of the fluorescence intensity.

When Figures 5 and 8 are compared, it is observed that the presence of melanin is underestimated by Figure 8, since only the intense pixels are attributed to melanin.

For each two-dimensional image of the three-dimensional stack, the calculation of the slopes of the logarithm of fluorescence intensity as a function of time corresponding to each pixel and the filtering of the corresponding two-dimensional mask are repeated in order to three-dimensionally visualize the associated melanin mask, as illustrated in Figure 7, and to characterize its distribution three-dimensionally throughout the epidermis, including in the stratum corneum.

The tests corresponding to Examples 2 and 3 which follow, carried out with this method, demonstrated differences in amount of melanin. Example 2 : evaluation of a treatment with dermocorticoids

The observations were carried out using a Dermalnspect^® microscope as previously described on the same area of skin before and after treatment with dermocorticoids.

Figure 9A shows a raw image of fluorescence of a sample of the area of skin before treatment and Figure 9C illustrates the corresponding melanin mask obtained by means of a method according to the invention.

The skin is treated with dermocorticoids (under occlusion).

Figures 9B and 9D illustrate a raw image of fluorescence of the sample and the corresponding melanin mask obtained by means of a method according to the invention after three weeks of treatment.

The method according to the invention allows specific detection of melanin; a comparison of the masks illustrated in Figures 8C and 8D demonstrates the decrease in the amount of melanin and thus allows better evaluation of the modifications induced by the dermocorticoids via three-dimensional quantification of the melanin in the skin.

Example 3 : characterization of human skin phototypes I to IV

Human skin is categorized into six phototypes according to its reaction to exposure to the sun. Dark skin (phototypes V and VI) has a greater amount of melanin which naturally screens UV rays. The method according to the invention allows a better analysis of the constituent pigmentation of phototypes I to IV corresponding to skin ranging from very fair to moderate brown.

Figures 10A and 10B show a raw image of fluorescence for skin samples for various phototypes and the corresponding melanin mask obtained by means of a method according to the invention.

On the basis of the masks of Figure 10B, the 3D density of melanin was calculated in order to obtain the comparative graph of Figure IOC demonstrating the increase in the amount of melanin with the phototype.

As illustrated by the mask of Figure 10B corresponding to phototype I, the method according to the invention makes it possible to quantify the presence of melanin, even at a low concentration. The method may thus be used for any type of tissue containing melanin.

Claims

1. Method for detection, quantification and/or visualization of an endogenous fluorophore, in a biological tissue, the method comprising the steps consisting in:

b) determining by processing these images, by performing a linear regression of the logarithm of the fluorescence signals, the spatial distribution in the sample of the slopes of said linear regression, and

c) generating information on the presence and/or the volume density or surface density of a fluorophore in the sample at least on the basis of this spatial distribution,

the fluorophore being melanin.

2. Method according to Claim 1 , the set of images comprising between three and five images

3. Method according to Claim 1 or 2, the integration of the fluorescence signal being carried out at regular intervals, in particular over a period of between 1 and 3 ns.

4. Method according to any one of the preceding claims, the information being generated after having eliminated the pixels having low-value slopes.

5. Method according to any one of the preceding claims, the information being generated in the form of at least one representation of the spatial distribution of melanin.

6. Method according to any one of the preceding claims, the information generated providing information on the surface and/or the volume taken up by melanin in the tissue.

7. Method according to any one of the preceding claims, which is implemented in vivo on a biological tissue.

8. Method according to any one of the preceding claims, the tissue being human skin, reconstructed or artificial skin, or melanized cells in culture.

9. Method for evaluating the action of a stimulus and/or of a treatment, which is in particular pro -pigmenting or depigmenting, on a biological tissue, comprising the steps consisting in:

evaluating the pigmentation in the tissue by means of the method according to any one of the preceding claims,

performing a second evaluation of the pigmentation in the tissue by means of a method according to any one of the preceding claims, the tissue having been exposed to an action of a stimulus chosen from: light radiation, in particular solar, ultraviolet (UV) or infrared (IR) radiation, a stimulus causing an inflammatory response, and a mechanical action, or a treatment, in particular with at least one pro -pigmenting or depigmenting cosmetic product, example lucinol or any other pigmentation-modulating chemical agent, comparing the information generated during the two evaluations, and evaluating the action of the stimulus or of the treatment on the pigmentation on the basis at least of this comparison.

10. Method for evaluating the action of a stimulus and/or of a treatment, which is in particular anti-pigmenting, pro -pigmenting or depigmenting, on a biological tissue, in which at least two regions of the tissue are exposed differently to the stimulus and/or treated differently, and in which the information on the presence and/or the amount of melanin in said regions, obtained by means of a method according to any one of claims 1 to 9, before and after exposure to said stimulus or to said treatment, are compared.