KR20140090184A - Non-invasive method for specific 3d detection, visualization and/or quantification of an endogenous fluorophore such as melanin in a biological tissue - Google Patents

Abstract

CLAIMS 1. A method for detecting, quantifying and / or visualizing an endogenous fluorophore in a living tissue comprising the steps of: a) after multiphoton excitation, for a time-dependent decrease in fluorescence signals in a sample Determining a spatial distribution in the sample of slopes of the linear regression by processing the images by performing a linear regression of the log of the fluorescent signals to obtain a set of successive three- or two- ; And c) generating information on the volume density or surface density and / or presence of the fluorophore in the sample based at least in part on this spatial distribution, wherein the fluorophore is melanin, Method for detection, quantification and / or visualization of red fluorescence ends.

Description

TECHNICAL FIELD [0001] The present invention relates to a non-invasive method for specific 3D detection, visualization and / or quantification of an endogenous fluorophore such as melanin in a living body tissue. BACKGROUND OF THE INVENTION [0002] BIOLOGICAL TISSUE}

The present invention relates to the observation of keratinous materials, such as skin, in living tissues.

More specifically, the present invention relates to a method aimed at determining the distribution of melanin in living tissues, in particular the nature and / or amount of distribution, in particular for evaluating the action of a cosmetic or therapeutic treatment, It is not limited.

The color of living tissues is closely related to the three-dimensional distribution and amount of melanin. Presently, characterization of the distribution and quantity of melanin is accomplished in two-dimensional form by quantification of melanin stained with Fontana Masson on white light imaging and histological sections, or in multiphoton imaging and image processing And can be carried out in a three-dimensional form by subsequent three-dimensional quantification of melanin.

Multiphoton microscopy facilitates deep observation of living tissues, especially highly-live living tissues. This is because infra-red light is less scattered and less absorbed, so that infrared light penetrates better into the tissues and generating signals using nonlinear mechanisms introduces stronger signal selection criteria in the scattering medium. This nonlinear mechanism involves the simultaneous interaction of two or even a few photons with the structure or molecule desired to be detected. Generally, an excitatory beam is focused and scanned in the sample, and the generated nonlinear signal is detected to obtain an image. The specificity of this kind of imaging is based on the optical part ability that imaging provides excitation power due to the nonlinear dependence of the signal. Since the excitation power density is very high at focus, the nonlinear effect under consideration occurs efficiently only at a limited focus volume; This intrinsic property ensures the suppression of the signal obtained in the sample, and thus enables obtaining a unique three-dimensional resolution on the order of micrometers. The imaging depth varies depending on the tissues, and the depth may be about 130 to 200 [mu] m for human skin.

In addition, due to the use of endogenous signal sources, such imaging techniques do not require labeling of tissue components. Fluorescence signals are produced by endogenous chromophores; In addition to melanin, NAD (P) H, flavins, keratin and elastin may be mentioned.

Application FR 2 944 425 discloses that based on the fact that on the skin at the level of the epidermal basal layer, highly enriched melanin is reflected by the fluorescence intensity stronger than that of other endogenous fluorophores, A method of evaluating skin pigmentation by microscopic examination is disclosed. Pixels with strong intensity are due to melanin. Nevertheless, this method is not always satisfactory because, firstly, it can be interrupted by other fluorophore groups which also express strong fluorescence intensities, such as keratin, and secondly, other endogenous fluorescent moieties Because it does not consider melanin, which expresses a fluorescent signal comparable to the fluorescence signal of the mouse.

A more specific method is to consider the fluorescence lifetime of melanin. In fact, the fluorescence lifetime of melanin is dependent on the intrinsic fluorescence properties of melanin and does not depend on the concentration of the fluorescent moiety in the excitation volume or on the intensity of the fluorescence. Melanin has a two-photon-excited fluorescence lifetime that is different from the fluorescence lifetimes of other endogenous fluorophore stages. This fluorescence lifetime can be measured by the method described in 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", vol. 77 (2011), pp 469-488, European Pharmacology and Biopharmaceutics Journal Based on images obtained by FLIM (Fluorescence Lifetime Imaging). JP2009-142597 describes a method for visualizing melanin by combining multiphoton microscopy and FLIM. The latter technique requires an integration of the fluorescence signal in time and space, an inexpensive operation in terms of time, in order to provide a good estimation of the fluorescence lifetime, and thus the possibility of application of this technique especially in the context of three- Limit.

Thus, there is a need to have the advantage of a sensitive, non-invasive method that is easy to implement, which allows for reliable, rapid and appropriate results for three-dimensional visualization of the distribution of melanin in living tissues.

It is an object of the present invention to develop a method which is simpler and faster than the prior art methods which makes it possible to detect melanin in particular and to characterize the three dimensional distribution of melanin in living tissue, .

It is also necessary to be able to verify the harmlessness or efficacy of the product, for example based on anti-pigmenting, depigmenting or pro-pigmenting actives used during processing have.

It may also be desirable to objectively differentiate the distribution and amount of melanin according to skin type, population, geographical location, or other dietary habits, and to determine the presence of actinic lentigenes, birthmarks (freckles) or stigma (masks sometimes appearing during pregnancy) , It is particularly advantageous to characterize pigmentation of the skin, in particular to characterize pigmentation of the skin.

In addition, in the context of an investigation to develop new tissues reproduced in a synthetic manner, it is necessary to objectify differences in the distribution and amount of melanin depending on the tissue model, particularly the type of skin model.

The present invention is intended to fulfill all or part of these needs.

Thus, an object of the present invention, in accordance with one aspect of the present invention, is a method for detecting, quantifying and / or visualizing an endogenous fluorophore component such as melanin in a living tissue,

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

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

and c) generating information about the amount and / or presence of the fluorophore in the sample based at least on the three-dimensional spatial distribution.

This method may be used in non-invasive, non-therapeutic, cosmetic contexts.

This method may make it possible to evaluate pigmentation.

The acquired images 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 representing the structure of the sample at a given instant and at a predetermined depth.

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

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

The value of the fluorescence signal of each pixel may be obtained by integration over a period of time of the two-photon excited fluorescence signal. The integration of the fluorescence signal can be carried out in particular 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 the reduction of the fluorescence signals can be obtained by linear regression of the log of fluorescence lifetimes. This method may require acquisition of only a limited number of three-dimensional temporal images, for example between three and five, suggesting a good compromise between acquisition time and precision. The use of slopes obtained by linear regression of the log facilitates limiting the number of such acquisitions.

Compared to the FLIM technology, the present invention reduces the number of temporal acquisitions and makes the implementation of the method easier and faster. While the FLIM technique allows a better estimation of fluorescence lifetime, but requires a significantly longer acquisition time (approximately 30 s) with degradation of the resolution of the three-dimensional image, the method according to the invention maintains resolution equivalent to the theoretical resolution of the microscope Dimensional visualization or quantification at the same time as it is performed.

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

The number of temporal acquisitions for each temporal pixel is, for example, equal to 4, and for each pixel, the two-photon excited fluorescence signal is at a regular interval of 2 ns, It is integrated according to progress.

The methods according to the present invention can be implemented in vivo, but can be implemented in ex vivo and in vitro samples.

The present invention improves the speed of acquisition methods and image processing methods. According to the present invention, 3D characterization of the tissue is possible and permits in vivo applications with high throughput.

The tissue may consist of human keratinous substances. The term "keratinous materials" is intended to mean, among others, hair, eyelashes, eyebrows, skin, nails, mucous membranes, scalp.

Biological tissues, in particular keratin materials according to the invention may be natural or artificial; The sample is, for example, human skin or reproduced or artificial skin.

The biological tissue may be melanized cells in a culture.

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

The generated information may provide information about the volume and / or surface occupied by melanin in the tissue and also its 3D distribution compared to other components of the tissue.

Another object of the present invention is to provide a method for evaluating the action of treatment and / or irritation on a living body tissue, in particular, prevention of pre-pigmentation, discoloration or pigmentation,

- evaluating pigmentation in living tissue by a method as described above,

- exposing the sample to a stimulus selected from light radiation, especially sun, ultraviolet (UVA and / or UVB) or infrared (IR) radiation, or in particular treatment with at least one precolouring, decolorizing or anti- , Exposing the sample to an inflammatory reaction, and causing mechanical action (tension, pressure, peeling, tear, exfoliation, polishing)

Performing a second evaluation of pigmentation in the living tissue by a method as described above,

- comparing the information generated during these two evaluations, and

At least evaluating the effect of treatment or stimulation on pigmentation based on this comparison.

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

The treatment may be selected 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 include exposing the body tissues to a treatment selected from application, injection, ingestion or inhalation of the product, in particular cosmetic products, or taking of food supplements and / or medicaments.

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

The product may have a decolorizing effect. Thus, the treatment may involve the application of any pigmentation-modifying chemical agent.

The method according to the present invention may also be used to evaluate the harmlessness or efficacy of decolorizing, anticoagulant or antioxidant active agents. The active agents, for example hydroquinone or retinoids, may be for therapeutic purposes and / or for cosmetic purposes.

The method according to the present invention may also be used to evaluate adverse effects of certain products, e. G. Deramocorticoids, on pigmentation.

According to another aspect of the present invention, an object of the present invention is to provide a method for evaluating the action of treatment and / or irritation, particularly, prevention of pigmentation, coloration or decolorization of biological tissue, Wherein at least two regions of the biotissue are exposed to different and / or different treatments of the stimulus, and before or after exposure to said treatment or said stimulation, of melanin in said areas, obtained by a method as previously described The information about the quantity and / or presence is compared.

In order to test the action of the product, the evaluation of the product after the treatment with the placebo and the evaluation after the treatment with the product may be compared.

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

Performing an evaluation of the distribution of melanin on samples of artificial or reproduced skin treated by a cosmetic compound or in living tissue of an individual and comparing the distribution of melanin in the same sample of artificial or reproducible skin Or the distribution of melanin in living tissues of the same individual.

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, lotion, ointment, oil, powder, and is not limited to this list.

The product may also be contained in biological tissues, in particular patches, dressings, bandages or masks applied to the skin of reproduced or artificial persons.

The product may not be a pigmentation or discoloration product intended to act only on the distribution and / or amount of melanin in living tissues. Thus, the product may have effects on the distribution and / or amount of melanin in living tissues, while at the same time, for example, to protect biological tissues, to provide moisture or to make up, Or to repair biological tissues. Thus, the product may contain other compounds, in particular other active agents than the active agents intended to act on the melanin of the biological tissues.

This method may make it possible to visualize melanin in living tissues; For example, it may be possible to know the distribution of melanin in the various layers of the epidermis of the skin.

Another object of the present invention according to another of the aspects of the present invention is a method for promoting treatment, particularly non-therapeutic treatment, wherein the action of treatment on melanin, as evidenced by the method as defined above, .

Another object of the present invention in accordance with another of the aspects of the present invention is to refer to the fact that the effectiveness of the product has been verified during the marketing of the product, for example in an advertisement or package, .

The present invention may be better understood by reading the following description of non-limiting examples of implementations of the invention and reviewing the drawings in the accompanying drawings.
Figure 1 shows the various steps of the method according to the invention.
Figure 2 illustrates and partially illustrates an example of a multiphotonic device that may be used in the method according to the present invention.
3 (a) to 3 (d) show examples of image acquisition according to the present invention.
Figures 4 (a) and 4 (b) illustrate various examples of temporal changes in fluorescence within a sample.
Figure 5 shows a parametric representation of fluorescence lifetimes for a portion of a skin sample at a fixed depth.
Figures 6 and 7 show two-dimensional and three-dimensional masks of the melanin distribution in skin samples, obtained by the method according to the invention.
Figure 8 shows a two-dimensional mask of melanin distribution in a skin sample, obtained by a method of the prior art.
Figures 9 (a) to 9 (d) show the amount of melanin in the skin sample before and after treatment.
Figures 10a, 10b and 10c show the characterization of human skin photo types I through IV by the amount of melanin.

Step 1 of the method according to the present invention may be to obtain sets of two-dimensional temporal images at various depths of a biopsy sample by multiphoton microscopy.

Each pixel may be obtained by integration over a time period of a two-photon excited fluorescence signal, for example, using a time-correlated single photon counting (TCSPC) photon counting device.

The use of a multiphoton microscopy device may allow automation of biopsy image acquisition steps.

A three-dimensional temporal image may be generated, in particular, from a stack of two-dimensional temporal images at various depths. Each stack may contain dozens of two-dimensional temporal images, for example 50 to 100 images, in particular 65 to 80 images.

In step 2, for each pixel of coordinates (x, y, z), the change in fluorescence intensity is studied as a function of time by calculating the log of fluorescence intensity. The log is then adjusted by linear regression to estimate the slope of the linear regression, which is related to fluorescence lifetime.

The acquired data may thus be represented as images of slopes obtained by linear regression of the log of fluorescence intensity.

A slope image, also called a parameter image, contains two types of pixels: pixels characterized by a low slope and pixels characterized by a high slope value. Pixels with high value slopes are due to melanin, which has a shorter lifespan compared to other endogenous fluorescent groups of epidermal cells, such as keratin, NAD (P) H, FAD,

In fact, melanin is characterized by two fluorescence lifetimes. One of the major fluorescence lifetimes is very short, about 0.1-0.2 ns (nanoseconds). Other fluorescence lifetimes of melanin are prime, between 0.7 and 1.4 ns, longer. In contrast, other endogenous fluorophores of epidermal cells, such as keratin, NAD (P) H, FAD, etc., have predominantly longer fluorescence lifetimes of greater than 1.5 ns.

Step 3 is to extract pixels corresponding to melanin from the slope image. Removing noise and and to detect melanin more specifically, to consider the size of the melanocytes cotton s (melanosomes), i.e., for it is a surface filter for comparing with the reference surface area of approximately 1 ㎛ ² For each of the 2 It is applied to the dimension image.

Information on the amount and / or presence of melanin in the sample is generated, for example, after eliminating fluorescence signals having structures with a size of less than 1 占 퐉 ² and low-valued slopes.

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 a melanin mask for one depth of the sample.

Step 2 of calculating the slopes and step 3 of filtering the associated mask may be performed on the entirety of the two-dimensional image corresponding to the predetermined depth and may be repeated on each image of the three-dimensional stack.

This method makes it possible to obtain a stack of two-dimensional representations that form a three-dimensional image of the melanin mask.

Based on the three dimensional melanin mask obtained by this method, melanin can be characterized by calculating the density of melanin and / or the spatial distribution of melanin.

Taking into account the speed of calculation, this method may be used in a screening method.

In combination with a system for measuring fluorescence lifetime, any known multiphoton microscopy system may be used to implement the method.

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 components of tissues.

Figure 2 illustrates and partially illustrates an example of a multi-photon device 100 that may be used in the method according to the present invention.

The photonic device 100 is, for example, be of the type developed by Jenlab DermaInspect ^® yarn.

The device 100 may be a femtosecond laser 10, such as a Ti: Sa laser, which may be tuned to the infrared wavelength range and provide pulses of about 100 femtoseconds at repetition rates on the order of 80 MHz. . The laser 10 emits an infrared beam 11 directed towards the laser beam scanning device 12 in the form of an "XY scanner ". The scanning 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 focused onto the keratin materials 13 by an objective lens 15. Thus, the two galvanometric mirrors allow scanning of the focus at a plane (x, y) perpendicular to the propagation direction (z-axis) of the beam.

The piezoelectric device makes it possible to translate the objective lens 15 and thus change the plane of focus to the keratinous materials 13. [ In this way, it is possible to reconstruct the three-dimensional distribution of melanin in keratinous materials by superimposition of the obtained images.

To do so, the signals produced at the focus may be detected thereafter, i.e., by epicollection through the excitation objective 15. The first dichroic mirror 14 may enable the selection of autofluorescence (AF) originating from the resulting multiphoton signals, in particular keratin materials 13.

The second dichroic mirror 16 may then be enabled to separate autofluorescence (AF) signals from other multi-photon signals, e.g., corresponding to a second harmonic generation (SHG). In any case, the signals pass through filters 17 and arrive at time-correlated single photon counting (TCSPC) 18, 19 to produce a combined image, for example an AF / SHG image Lt; / RTI >

The first and second detectors 18 and 19 may be enabled to generate signals 18a and 19a that are transmitted to the electronic device 20 for signal acquisition and processing.

Example 1: In vivo observation of human skin

During the test, the three-dimensional image in the body on the people's arm to were obtained in succession, one of the three-dimensional image, 40x / 1.3 NA with excitation wavelength of an objective lens and 760 nm,, skin that is obtained by using the DermaInspect ^® device Lt; RTI ID = 0.0 > 2 < / RTI >

Figures 3 (a) - (d) show only four two-dimensional temporal images obtained in vivo on the skin of the forearm of a healthy volunteer near the base layer of the epidermis at a depth of 50 [mu] m against the surface of the skin Which represents a temporal sequence of three-dimensional images. For each temporal image, the fluorescence signal was integrated over a period of 2 ns.

Under experimental conditions, for a point of a sample containing melanin, a plurality of emitted fluorescent photons are collected during a first temporal acquisition. The greater the concentration of melanin, the higher the number of photons of this first acquisition. An arrow I in FIG. 3 (a) corresponding to the first temporal channel represents a zone of high melanin concentration based on the intensity criterion. At this level, the highly enriched melanin produces a fluorescence signal with a stronger intensity than the intensity of other endogenous fluorophore.

Figures 4 (a) and 4 (b) show the temporal evolution curves (Figure 4 (a)) for pixels corresponding to points of the sample with melanin and for pixels corresponding to points of the sample without melanin, And the adjustment of the logarithm of the fluorescence intensity by linear regression (Fig. 4 (b)).

Figures 5, 6, and 8 correspond to two-dimensional representations of human skin at a depth of 50 [mu] m. Figure 5 shows a parameter image of the slopes obtained by linear regression of the log of fluorescence intensity. The image has two kinds of pixels, which are attributed to melanin for the reasons described above: pixels characterized by a low slope, and pixels characterized by a high slope value.

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

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

For each two-dimensional image of the three-dimensional stack, as shown in Fig. 7, three-dimensionally visualizing the associated melanin mask and characterizing its distribution three-dimensionally across the skin, including the horny layer The calculation of the slopes of the log of fluorescence intensity as a function of time corresponding to each pixel and the filtering of the corresponding two-dimensional mask are repeated.

The tests corresponding to the subsequent Examples 2 and 3 conducted in this way demonstrated the difference in the amount of melanin.

Example 2: Dermo Corticoids  Evaluation of treatment by

Observations were made using the DermaInspect ^® microscope as described above for the same areas of skin before and after treatment with the dermacocorticoids.

Figure 9 (a) shows a raw image of the fluorescence of a sample of the skin zone before treatment, and Figure 9 (c) shows the corresponding melanin mask obtained by the method according to the invention.

The skin is treated (with occlusion) with democorticoids.

Figures 9 (b) and (d) show the original image of the fluorescence of the sample after three weeks treatment and the corresponding melanin mask obtained by the method according to the invention.

The method according to the invention allows a specific detection of melanin; Comparisons of the masks shown in Figures 8 (c) and 8 (d) demonstrate a reduction in the amount of melanin and thus a better evaluation of the modifications caused by the dermacocorticoids by three-dimensional quantification of melanin in the skin Allow.

Example 3: Human skin Photo types  I- IV Characterization

Human skin is classified into six phototypes depending on the reaction of the skin when exposed to the sun. Darkish skin (phototypes V and VI) have a higher amount of melanin that innately screens UV rays. The method according to the invention allows a better analysis of the constituent pigmentation of the phototypes I to IV corresponding to the skin, from very fair to moderately brown.

Figures 10A and 10B show the original image of the fluorescence for skin samples for various photo types and the corresponding melanin mask obtained by the method according to the present invention.

Based on the masks in FIG. 10B, the 3D density of melanin was calculated to obtain the comparative graph of FIG. 10C, which demonstrates an increase in the amount of melanin by the phototype.

As shown by the mask of Fig. 10B corresponding to Photo Type I, the method according to the invention makes it possible to quantify the presence of melanin even at low concentrations. The method may therefore be used for any type of tissue containing melanin.

Claims (10)

A method for detecting, quantifying and / or visualizing an endogenous fluorophore in a living tissue,
a) obtaining, after multiphoton excitation, a set of successive three-dimensional or two-dimensional images providing information on a decrease over time of the fluorescence signals in the sample,
b) determining a spatial distribution in the sample of slopes of the linear regression by performing a linear regression of the log of the fluorescent signals and processing the images; and
c) generating information on the volume density or the surface density and / or the presence of said fluorescent dots in said sample based at least in part on this spatial distribution,
Wherein the fluorophore is melanin, for detection, quantification and / or visualization of an endogenous fluorophore in a living tissue. The method according to claim 1,
Wherein the set of images comprises between three and five images. ≪ Desc / Clms Page number 19 > 3. The method according to claim 1 or 2,
Method for detection, quantification and / or visualization of endogenous fluorophore in living tissue, wherein the integration of said fluorescence signal is carried out over regular intervals, in particular between 1 and 3 ns. 4. The method according to any one of claims 1 to 3,
Quantifying and / or visualizing an endogenous fluorophore in a living tissue, wherein the information is generated after removing pixels with low value slopes. 5. The method according to any one of claims 1 to 4,
Wherein the information is generated in the form of at least one representation of the spatial distribution of melanin, for detection, quantification and / or visualization of an endogenous fluorophore in a living tissue. 6. The method according to any one of claims 1 to 5,
Wherein the information provided provides information about the volume and / or surface occupied by melanin in the living tissue, for detection, quantification and / or visualization of an endogenous fluorophore in a living tissue. 7. The method according to any one of claims 1 to 6,
A method for detection, quantification and / or visualization of an endogenous fluorophore in a living body tissue, said in vivo implementation of said biopsy. 8. The method according to any one of claims 1 to 7,
A method for detection, quantification and / or visualization of an endogenous fluorophore in a living tissue, wherein the biological tissue is human skin, reproduced or artificial skin, or melanized cells in a culture. 1. A method for evaluating the action of treatment and / or stimulation on living tissue, especially pro-pigmenting or depigmenting,
- evaluating pigmentation in the biotissue by the method of any one of claims 1 to 8,
- Performing a second evaluation of pigmentation in the biotissue by the method according to any one of claims 1 to 8, wherein the biotissue is exposed to light radiation, in particular the sun, ultraviolet (UV) or Is exposed to the action of the stimulus selected from infrared (IR) radiation, or in particular by treatment with at least one preochromin or bleaching cosmetic product, e.g. lucinol, or any other pigmentation-modifying chemical agonist, And performing a second evaluation of the pigment deposition, causing a mechanical action,
- comparing the information generated during these two evaluations, and
- evaluating the action of the treatment or stimulation on the pigmentation based at least on this comparison, in particular the method of evaluating the action of treatment and / or irritation to the biotissue, in particular pre-pigmentation or decolorization. As a method for evaluating the action of treatment and / or irritation on living tissue, particularly anti-pigmenting, total pigmentation or decolorization,
The method according to any one of claims 1 to 9, wherein at least two regions of the biotissue are exposed and / or treated differently to the stimulus, and before or after the treatment or exposure to the stimulus And / or the presence of melanin in the areas, compared to information obtained about the presence and / or presence of melanin in the areas, wherein the information about the amount and / or presence of melanin in the areas is compared Way.

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