JP6147756B2 - Non-invasive method for specific 3D detection, visualization and / or quantification of endogenous phosphors such as melanin in living tissue - Google Patents

Description

  The present invention relates to observation of keratin materials such as living tissue, particularly skin.

  The present invention is more particularly directed to determining the distribution of melanin in biological tissue, in particular the amount and / or nature of the distribution, with the particular aim of assessing the effect of a cosmetic or therapeutic treatment. However, the present invention is not limited to this.

  The color of living tissue is closely related to the amount and three-dimensional distribution of melanin. Today, the characterization of the amount and distribution of melanin is by two-dimensional morphology with white light imaging and quantification of melanin with tissue sections stained with Fontanamason, or multiphoton imaging and three-dimensional quantification of melanin after image processing It can be implemented in any of the three dimensional forms.

  Multiphoton microscopy facilitates deep observation of living tissue, particularly living tissue that is highly scattered. This is because the infrared light is less scattered and less absorbed, so the infrared light is better transmitted into the tissue and a stronger signal selection criterion is scattered by using a non-linear mechanism to generate the signal. This is because it is introduced into the medium. In this nonlinear mechanism, two or even several photons interact with the molecule or structure to be detected simultaneously. In general, the excitation beam is focused and scanned in the sample, and the generated non-linear signal is detected to obtain an image. The peculiarity of this type of image is based on the optical section capacity obtained due to the non-linear dependence of the signal with the excitation output. Since the excitation power density is very high at the focal point, the nonlinear effect under consideration is only exerted efficiently in a limited focal volume, and this intrinsic property ensures the containment of the signal obtained in the sample. Therefore, it is possible to obtain a unique three-dimensional resolution of the order of micrometers. The imaging depth varies depending on the tissue and may be about 130-200 μm for human skin.

  Furthermore, due to the use of endogenous signal sources, this imaging technique does not require the labeling of tissue components. The fluorescent signal is generated by the endogenous chromophore and can include NAD (P) H, flavin, keratin, and elastin in addition to melanin.

  Patent Document 1 is based on the fact that the presence of a high concentration of melanin at the epidermal basal layer level of the skin is reflected by the fact that there is a stronger fluorescence intensity than in the case of other endogenous phosphors. A method for assessing skin pigmentation by photon microscopy is disclosed. Pixels with strong intensity are due to melanin. Nonetheless, this method can be first disturbed by other phosphors that also exhibit strong fluorescence intensity, such as keratin, and secondly, comparable to that of other endogenous phosphors. Since melanin showing a fluorescent signal is not taken into account, it is not always satisfactory.

  A more specific method is to consider the fluorescence lifetime of melanin. In fact, the fluorescence lifetime of melanin depends on the intrinsic fluorescence properties of melanin and does not depend on the fluorescence intensity in the excitation volume or the concentration of the phosphor. Melanin has a two-photon excited fluorescence lifetime that is different from that of other endogenous phosphors. This fluorescence lifetime can be estimated based on images acquired by FLIM (Fluorescence Lifetime Imaging). This technique is described in Non-Patent Document 1, and the fluorescence signal decreases with time. Is to monitor. U.S. Patent No. 6,057,032 describes a method for visualizing melanin that combines multiphoton microscopy and FLIM. The latter technique requires integration of the fluorescence signal in time and space, i.e. expensive computations with respect to time, in order to make a good estimate of the fluorescence lifetime, thereby applying this technique especially in the context of three-dimensional imaging. The possibility to do is limited.

French Patent Application No. 2944425 Specification JP 2009-142597 A

M.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)

  Therefore, a sensitive and non-invasive method that is easy to implement for three-dimensional visualization of the distribution of melanin in living tissue, which makes it possible to obtain reliable, quick and relevant results. You need to have a profit.

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

  There is also a need to be able to verify the efficacy or harmlessness of products used during treatment, for example based on anti-pigmentation, depigmentation, or pigmentation-promoting active agents.

  To objectively differentiate the amount and distribution of melanin depending on the type of skin, population, geographical location, or other diet, etc., and also with photo-induced moles, freckles, or melasma (during pregnancy) It is also advantageous to characterize skin pigmentation, in particular to characterize skin pigmentation disorders, such as masks that sometimes appear.

  Furthermore, in the context of exploration to develop new tissues that are reconstructed by synthesis, it is required to make objective differences in the amount and distribution of melanin depending on the type of tissue model, particularly the skin model.

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

The subject of the present invention is therefore, according to one of its aspects, a method for detecting, quantifying and / or visualizing endogenous phosphor components such as melanin in biological tissue:
a) obtaining a set of continuous three-dimensional or two-dimensional images that provide information about the decay of the fluorescence signal in the sample over time after multiphoton excitation;
b) determining the spatial distribution in the sample of the slope of the linear regression by processing these images by performing a linear regression of the logarithm of the fluorescence signal;
c) generating information on the presence and / or amount of phosphor in the sample based at least on this three-dimensional spatial distribution.

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

  The method can allow assessment of pigmentation.

  The acquired image is a three-dimensional temporal image. The term “three-dimensional temporal image” is intended to mean a series of consecutive three-dimensional images over time.

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

  Each pixel of each three-dimensional or two-dimensional image may be defined by spatial coordinates (x, y, z) where z is the depth of the sample. Each two-dimensional image thus represents a specific case of a three-dimensional image in which acquisition is performed only at a given depth z.

  The pixels of a three-dimensional image are grouped together into sets of pixels having the same spatial coordinates of the image obtained at various moments, and these sets of pixels are called “temporal pixels”.

  The value of the fluorescence signal of each pixel may be obtained by integrating the two-photon excitation fluorescence signal with respect to a certain time. 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 with a period equal to 1, 1.5, 2, 2.5 or 3 ns.

  The slope of the decrease in fluorescence signal can be obtained by linear regression of the logarithm of fluorescence lifetime. The method may only require acquisition of a limited number of three-dimensional temporal images, for example, between 3 and 5 images that are well balanced between acquisition time and accuracy. The use of the slope obtained by logarithmic linear regression facilitates this limitation of the number of acquisitions.

  Compared to FLIM technology, the present invention reduces the number of time acquisitions and makes the implementation of the method easier and faster. The FLIM technology allows better estimation of fluorescence lifetime, but requires a very long acquisition time (approximately 30 seconds) with degraded resolution of the 3D image, and the method according to the present invention is faster. And is particularly advantageous for obtaining three-dimensional visualization or quantification, while retaining a resolution equivalent to the theoretical resolution of the microscope.

  The total acquisition time for a temporal pixel at the coordinates (x, y, z) of a given sample is about 0.28 μs instead of being several ms with FLIM.

  The number of temporal acquisitions for each temporal pixel is, for example, equal to four, and for each pixel, the two-photon excitation fluorescence signal is integrated over time between two acquisitions, especially at regular intervals of 2 ns. Is done.

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

  The present invention improves the speed of acquisition and image processing methods. The present invention enables 3D characterization of tissues and enables in vivo applications with high throughput.

  The tissue may be composed of human keratin material. The term “keratin material” is intended to mean in particular the hair, eyelashes, eyebrows, skin, nails, mucous membranes, scalp.

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

  The living tissue may be a cell that has been melanized during 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 surface and / or volume occupied by melanin in the tissue, and information about its 3D distribution compared to other components of the tissue.

The subject of the present invention, according to another of its aspects, is a method for assessing the effect on a living tissue of a stimulus and / or treatment, in particular pigmentation promotion, depigmentation, or anti-pigmentation. There:
-Assessing pigmentation of biological tissue using the method described above;
-Samples: from light radiation, especially sunlight, ultraviolet (UVA and / or UVB), or infrared (IR) radiation, stimuli that cause an inflammatory response, and mechanical action (tensile, pressure, delamination, dissociation, shedding, wear) Exposure to selected stimuli or in particular treatment with at least one pigmentation, depigmentation or anti-pigmentation product;
-Performing a second assessment of pigmentation of biological tissue using the method described above;
-Comparing the information generated during the two evaluations;
-Assessing the effect of the stimulus or treatment on the pigmentation based at least on this comparison.

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

  The treatment may be selected from: depositing, injecting, ingesting or inhaling a product, especially a cosmetic product.

  Treatment may correspond to intake of dietary supplements and / or pharmaceuticals. The treatment may comprise exposing the biological tissue to a treatment selected from: the attachment, infusion, ingestion, or inhalation of a product, in particular a cosmetic, or ingestion of dietary supplements and / or pharmaceuticals.

  The term “cosmetics” shall mean the product defined in Directive 93/35 / EEC, June 14, 1993, Amendment Directive 76/768 / EEC.

  The product may have a depigmenting action. Thus, the treatment may include the attachment of any pigmentation modifying chemical.

  The method according to the invention may be used to assess the efficacy or harmlessness of a depigmentation, anti-pigmentation, or pigmentation promoting active agent. The active agent may be for therapeutic purposes, for example hydroquinone or retinoid and / or for cosmetic purposes.

  The method according to the present invention may be used to assess side effects on pigmentation of certain products, such as skin corticoids.

  The subject of the present invention, according to another of its aspects, is a method for assessing the effect on a living tissue of a stimulus and / or treatment, in particular anti-pigmentation, promotion of pigmentation or depigmentation. Wherein at least two regions of tissue are exposed to a stimulus in different states and / or treated in a different state of the region obtained using the method described above before and after exposure to the stimulus or the treatment. A method in which information regarding the presence and / or amount of melanin is compared.

  To test the effect of the product, the assessment made after treatment of the product with a placebo may be compared to the assessment made after treatment with the product.

  A placebo is, for example, the same cosmetic medium as that of a product used for treatment, but does not contain a corresponding active agent.

  An assessment of the distribution of melanin in a sample of living tissue or artificial or reconstructed skin of an individual treated with a cosmetic compound, and the same tissue or artificial or reconstructed skin of the same individual treated with a cosmetic compound placebo It is possible to compare with an assessment of the distribution of melanin in the same sample.

  The treatment may correspond to the attachment of a product, in particular a cosmetic product. The product may take the form of creams, lotions, ointments, oils, powders, for example, and this list is not limiting.

  The product may be contained in a carrier, such as a patch, dressing, bandage, or mask, that is attached to living tissue, particularly reconstructed or artificial skin.

  The product may not be a pigmented or depigmented product that is only intended to affect the amount and / or distribution of melanin in living tissue. The product thus influences the amount and / or distribution of melanin in the living tissue, for example at the same time as making up, moistening or protecting the living tissue, in particular protecting it from sunlight, or repairing the living tissue. May be intended. Thus, the product may contain various compounds, in particular active agents other than active agents intended to act on melanin in living tissues.

  The method may allow visualization of melanin in living tissue, for example, it may allow to know the distribution of melanin in various layers of the skin epidermis.

  Another subject of the invention refers, according to another of its aspects, to treatment, in particular non-therapeutic treatment, the effect of treatment on melanin demonstrated using the method defined above. It is a way to facilitate treatment.

  Another subject of the invention, according to another of its aspects, is the fact that the efficacy of the product has been verified using the method defined above during the sale of the product, for example in advertising or on packaging. A method comprising the steps of referencing.

  The invention can be better understood from reading the following description of non-limiting examples in which it is implemented and from examining the figures in the accompanying drawings.

FIG. 3 shows the various steps of the method according to the invention. FIG. 2 schematically and partially represents an example of a multiphoton device that may be used in the method according to the invention. It is a figure which shows the example of the image acquisition by this invention. It is a figure which shows the example of the image acquisition by this invention. It is a figure which shows the example of the image acquisition by this invention. It is a figure which shows the example of the image acquisition by this invention. It is a figure which shows the various examples of the time change of the fluorescence in a sample. It is a figure which shows the various examples of the time change of the fluorescence in a sample. FIG. 6 shows a parametric display of fluorescence lifetime for a section of a fixed depth skin sample. FIG. 3 shows a two-dimensional mask of melanin distribution in a skin sample obtained using the method according to the invention. FIG. 3 shows a three-dimensional mask of melanin distribution in a skin sample obtained using the method according to the invention. It is a figure which shows the two-dimensional mask of the melanin distribution in the skin sample obtained using the method of a prior art. FIG. 3 shows the amount of melanin in a skin sample before and after treatment. FIG. 3 shows the amount of melanin in a skin sample before and after treatment. FIG. 3 shows the amount of melanin in a skin sample before and after treatment. FIG. 3 shows the amount of melanin in a skin sample before and after treatment. FIG. 4 shows the characterization of human skin phototypes I to IV according to the amount of melanin. FIG. 4 shows the characterization of human skin phototypes I to IV according to the amount of melanin. FIG. 4 shows the characterization of human skin phototypes I to IV according to the amount of melanin.

  Step 1 of the method according to the invention may be in the acquisition of several sets of two-dimensional temporal images at various depths of a biological tissue sample by multiphoton microscopy.

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

  Use of a multi-photon microscopy device can allow automation of the tissue image acquisition step.

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

  In step 2, for each pixel of coordinates (x, y, z), the change in fluorescence intensity as a function of time is investigated by calculating its logarithm. The log is subsequently adjusted using linear regression to estimate its slope associated with fluorescence lifetime.

  Thus, the data obtained can be represented by a gradient image obtained by logarithmic linear regression of fluorescence intensity.

  Gradient images, also called parametric images, contain two types of pixels: pixels characterized by low gradients and pixels characterized by high gradient values. Pixels with high value gradients are due to melanin, which has a short lifetime compared to other endogenous phosphors of epidermal cells such as keratin, NAD (P) H, FAD.

  In fact, melanin is characterized by two fluorescence lifetimes. The main one is very short, about 0.1 to 0.2 ns (nanoseconds). Another fluorescence lifetime of melanin is less and longer, between 0.7 and 1.4 ns. By comparison, other endogenous phosphors of epidermal cells such as keratin, NAD (P) H, FAD have primarily longer fluorescence lifetimes and are longer than 1.5 ns.

Step 3 is to extract pixels corresponding to melanin from the gradient image. Surface filtering is applied to each two-dimensional image, for example, taking into account the size of the melanosomes, ie compared to a reference surface area of about 1 μm ² in order to remove noise and more specifically detect melanin.

Information regarding the presence and / or amount of melanin in the sample is generated, for example, after eliminating fluorescent signals with low values of slope and structures with a size of less than 1 μm ² .

  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 at one depth of the sample.

  Step 2 of calculating the gradient and step 3 of filtering the relevant mask may be performed on the entire two-dimensional image corresponding to a given depth and may be repeated for each image in the three-dimensional stack.

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

  Based on the three-dimensional melanin mask obtained using this method, it is possible to characterize melanin by calculating its density and / or its spatial distribution.

  If the calculation is quick, this method may be used for screening methods.

  Any known multiphoton microscope system in combination with a system for measuring fluorescence lifetime may be used to perform the method.

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

  FIG. 2 schematically and partly represents an example of a multiphoton device 100 that may be used in the method according to the invention.

  The multiphoton device 100 is, for example, of the DermaInspect (registered trademark) type developed by Jenlab.

  Device 100 is a femtosecond laser 10, such as a titanium-sapphire (Ti: Sa) laser, that can be tuned to the infrared wavelength range and can provide pulses of about 100 femtoseconds at a repetition rate of about 80 MHz. Including. The laser 10 emits an infrared beam 11 directed to a laser beam scanning device 12 in the form of an “XY scanner”. The scanning of the laser beam can be obtained by angular movement of the two galvanometric mirrors of the scanning device 12. The beam is then reflected by the dichroic mirror 14 and focused onto the keratin material 13 using the objective lens 15. Therefore, the two galvanometric mirrors enable scanning of the focal point in a plane (x, y) orthogonal to the beam propagation direction (z-axis).

  The piezoelectric device makes it possible to translate the objective lens 15 and thus to change the plane focused by the keratin material 13. In this way, it is possible to reconstruct the three-dimensional distribution of melanin in the keratin material by superimposing the acquired images.

  To do so, the signal generated at the focal point may then be detected, i.e. detected by epi-collection through the excitation objective 15. The first dichroic mirror 14 allows the selection of the generated multiphoton signal, in particular the autofluorescence (AF) derived from the keratin material 13.

  The second dichroic mirror 16 then allows the separation of the autofluorescence (AF) signal from other multiphoton signals, eg corresponding to second harmonic generation (SHG). In any case, the signal passes through the spectral filter 17 and reaches a photon counting detector (TCSPC) 18, 19 and thus a combined image, eg AF / SHG images can be generated.

  The first and second detectors 18 and 19 enable the generation of signals 18a and 19a that are transmitted to the electronic device 20 for signal acquisition and processing.

In vivo observation of human skin During the study, three-dimensional images were obtained continuously in vivo with respect to the human forearm, one single three-dimensional image with an excitation wavelength of 760 nm and a 40x / 1.3 NA objective. It consisted of a stack containing 70 two-dimensional images obtained using a DermaInspect® device, acquired at a depth ranging from 2.346 μm from the surface of the skin.

  FIGS. 3A-3D show three-dimensional images in chronological order, obtained in vivo on the forearm skin of healthy volunteers, close to the basal layer of the epidermis at a depth of 50 μm from the surface of the skin. Only two-dimensional temporal images are shown. For each temporal image, the fluorescence signal was integrated over a time of 2 ns.

  Under experimental conditions, with respect to sample points containing melanin, the majority of the emitted fluorescent photons are collected during the initial time acquisition. The higher the concentration of melanin, the greater the number of photons from this initial acquisition. The arrow I in FIG. 3A, corresponding to the first temporal channel, shows an area of high melanin concentration based on intensity criteria. At this level, high concentrations of melanin produce a fluorescent signal with a stronger intensity than is the case with other endogenous phosphors.

  FIGS. 4A and 4B show the temporal generation curve (FIG. 4A) and fluorescence using linear regression for image pixels corresponding to sample points containing melanin and pixels corresponding to sample points not containing melanin. An example with logarithmic adjustment of intensity (FIG. 4B) is shown.

  5, 6 and 8 correspond to a two-dimensional representation of human skin at a depth of 50 μm. FIG. 5 shows a parametric image of the slope obtained by logarithmic linear regression of fluorescence intensity. The image has two types of pixels: those characterized by a low gradient and those characterized by a high gradient value, due to melanin for the reasons described above.

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

  When comparing FIG. 5 and FIG. 8, it is observed that the presence of melanin is underestimated by FIG. 8, because only strong pixels are attributed to melanin.

  For each two-dimensional image of the three-dimensional stack, the calculation of the logarithmic gradient of the fluorescence intensity as a function of time corresponding to each pixel and the filtering of the corresponding two-dimensional mask are repeated, as shown in FIG. The relevant melanin mask is visualized three-dimensionally and its distribution is characterized three-dimensionally throughout the epidermis including the stratum corneum.

  The following tests corresponding to Example 2 and Example 3 were carried out by this method and demonstrated differences in the amount of melanin.

Evaluation of treatment with cutaneous corticoids Observations were performed on the same area of skin before and after treatment with cutaneous corticoids using the aforementioned DermaInspect® microscope.

  FIG. 9A shows a raw fluorescent image of a sample of the area of skin prior to treatment, and FIG. 9C shows the corresponding melanin mask obtained using the method according to the invention.

  The skin is treated with a dermal corticoid (in the closed state).

  FIG. 9B and FIG. 9D show a raw fluorescence image of the sample after 3 weeks of treatment and the corresponding melanin mask obtained using the method according to the invention.

  The method of the present invention allows for the specific detection of melanin, and the comparison of the masks shown in FIGS. 8C and 8D demonstrates a reduction in the amount of melanin, and thus the three-dimensional quantification of melanin in the skin A better assessment of modifications induced by dermal corticoids via

Characterization of human skin phototypes I to IV Human skin is classified into six phototypes according to their response when exposed to sunlight. Dark skin (phototypes V and VI) has more melanin and naturally screens for UV light. 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 white to mild brown.

  FIGS. 10A and 10B show raw fluorescent images for various phototype skin samples and corresponding melanin masks obtained using the method according to the invention.

  Based on the mask of FIG. 10B, the 3D density of melanin was calculated to obtain the comparative graph of FIG. 10C demonstrating an increased amount of melanin with phototype.

  As shown by the mask of FIG. 10B corresponding to phototype I, the method according to the invention allows the quantification of the presence of melanin even at low concentrations. Thus, the method can be used on any type of tissue containing melanin.

DESCRIPTION OF SYMBOLS 10 Femtosecond laser 11 Infrared beam 12 Scanning device 13 Keratin material 14 1st dichroic mirror 15 Objective lens 16 2nd dichroic mirror 17 Spectral filter 18 Photon counting detector 19 Photon counting detector 20 Electronic device 100 Multiphoton device

Claims (9)

A method for detecting, quantifying, and / or visualizing endogenous phosphors in biological tissue comprising:
a) obtaining a set of continuous three-dimensional or two-dimensional images that provide information about the decay of the fluorescence signal in the sample over time after multiphoton excitation;
b) determining the spatial distribution in the sample of the slope of the linear regression by processing these images by performing a linear regression of the logarithm of the fluorescence signal;
c) generating information on the presence and / or volume density or surface density of the phosphor in the sample based at least on this spatial distribution;
The phosphor is Ri melanin der,
The method wherein the information is generated after excluding pixels with low value gradients .   The method of claim 1, wherein the set of images includes between 3 and 5 images.   The method according to claim 1 or 2, wherein the integration of the fluorescence signal is performed at regular intervals, in particular for a period between 1 ns and 3 ns. Wherein the information is generated by at least one display in the form of the spatial distribution of melanin, a method according to any one of claims 1 to 3. 5. A method according to any one of claims 1 to 4 , wherein the information generated provides information regarding the surface and / or volume occupied by melanin in the tissue. On living tissue, it is carried out in the in vivo, the method according to any one of claims 1 to 5. 6. The method according to any one of claims 1 to 5 , wherein the tissue is human skin, reconstituted or artificial skin, or cells melanized in culture. A method for assessing the effect on a living tissue of a stimulus and / or treatment, in particular pigmentation promotion or depigmentation, comprising:
Detecting the fluorescent material in said tissue using the method according to any one of claims 1 to 7, by quantifying, and / or visualization, the generating the information about the pigmentation of the tissue 1 step ,
Detecting the fluorescent material in said tissue using the method according to any one of claims 1 to 7, by quantifying, and / or visualization, the generating the information about the pigmentation of the tissue Two steps , wherein the tissue is selected from light radiation, in particular sunlight, ultraviolet (UV) or infrared (IR) radiation, a stimulus causing an inflammatory response, and a mechanical action, or in particular at least one kind of pigmentation promoting or depigmentation of cosmetics, for example lucinol or those exposed to the action of treatment with any other pigmentation regulatory chemicals, comprising the steps,
Method comprising the step of comparing the information generated in said first step and said second step. A method for assessing the effect on living tissue of stimuli and / or treatments, in particular antipigmentation, pigmentation promotion or depigmentation, comprising:
Detecting, quantifying, and / or phosphors in at least two regions of the tissue prior to exposure to the stimulation and / or treatment using the method of any one of claims 1-7. Or generating, by visualizing, information regarding the presence and / or amount of melanin in the tissue;
Detecting and quantifying phosphors in at least two regions of the tissue that have been exposed to the stimulus and / or treatment in different states using the method of any one of claims 1-7. Generating information relating to the presence and / or amount of melanin in at least two regions of the tissue by visualization and / or visualization;
Comparing the information generated during the first step and the second step .