CN103930767B - The noninvasive method of specific 3D detections, visualization and/or the quantization rolled into a ball in biological tissue such as the Intrinsic fluorescence of melanin - Google Patents
The noninvasive method of specific 3D detections, visualization and/or the quantization rolled into a ball in biological tissue such as the Intrinsic fluorescence of melanin Download PDFInfo
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Abstract
The present invention relates to a kind of detection, quantization and/or the visualization method of the Intrinsic fluorescence group being used in biological tissue, it the described method comprises the following steps：A) after multiphoton excitation, one group of continuous 3-D view or two dimensional image are gathered, the image provides the information weakened on the fluorescence signal in sample with the time；B) carry out linear regression to handle these images by the logarithm to the fluorescence signal, determine the spatial distribution of the slope of the linear regression in the sample；And this spatial distribution c) is at least based on, the information on fluorogen presence in the sample and/or bulk density or superficial density is generated, the fluorogen is melanin.
Description
Technical field
The present invention relates to biological tissue, the observation of the especially keratin material of such as skin.
The present invention more specifically but not exclusively to be intended to determine biological tissue in melanin distribution, be especially distributed
Quantity and/or property, especially for assess cosmetic treatments or treatment processing effect.
Background technology
The color of biological tissue and the quantity of melanin and distributed in three dimensions have close relation.Nowadays, with two dimensional form, lead to
Cross the white light imaging of the melanin in Histological section using Fontana Masson dyeing and quantify, or with three-dimensional shaped
Formula, after image processing, by the imaging of the multi-photon of melanin and 3-D quantitative, can carry out characterizing melanin quantity and
Distribution.
Multiphoton microscope inspection promotes the deep observation of biological tissue, the especially tissue of the work of height scattering.This is
Because being absorbed due to infrared light by less scattering and by less, therefore infrared light is preferably penetrated into tissue, and utilizes non-thread
Property mechanism introduces stronger signal behavior standard to produce signal in scattering medium.This Nonlinear Mechanism be related to two kinds or
Interacted while molecule or structure that even more kinds of photons and expectation are detected.Generally, excitation beam is focused and scanned
In the sample, formed nonlinear properties are detected, to obtain image.It is non-limiting due to the signal with exciting power
Rely on, the particularity of such imaging is based on the optical section performance provided.Because exciting power density is in focus
Place is very high, therefore the nonlinear effect considered is only effectively appeared in limited volume of focus；This basic performance
The control of the signal obtained in the sample is ensure that, therefore the dimensional resolution of intrinsic about micron can be obtained.Imaging
Depth changes according to tissue, for human skin, and imaging depth about can be 130 μm to 200 μm.
Further, since using endogenous signal source, this imaging technique does not need tagged tissue composition.Given birth to by endogenous
Color group, forms fluorescence signal；Except melanin, it can be mentioned that NAD (P) H (nicotinamide-adenine dinucleotide phosphate), flavine,
Keratin and elastin laminin.
Application FR 2 944 425 discloses a kind of method by multiphoton microscope inspection and evaluation cutaneous pigmentation,
This method based in skin, epidermis basalis level, it is strong by rolling into a ball stronger fluorescence than other Intrinsic fluorescences
Spend to reflect the melanin of high concentration.Pixel with high intensity is attributed to melanin.However, this method is not always made us
Satisfied, because, first, this method can be by other fluorogen (e.g., keratin) institutes for also showing that high fluorescent
Upset, secondly, this method does not consider the melanin with the fluorescence signal that can be mentioned in the same breath with other Intrinsic fluorescence groups.
More specific method includes the fluorescence lifetime for considering melanin.Really, the fluorescence lifetime of melanin is depended on
The primary fluorescence property of melanin, is not dependent on fluorescence intensity and the fluorogen concentration in excitation volume.Melanin has double
Photon excitation fluorescence lifetime, the fluorescence lifetime is different from the fluorescence lifetime that other Intrinsic fluorescences are rolled into a ball.According to passing through FLIM (fluorescence
Life-span is imaged) image that is obtained, this fluorescence lifetime can be estimated, the FLIM is the article 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 " (European Journal of Pharmaceutics and Biopharmaceutics, 2011 the 77th
Volume, page 469 to page 488) described in including a kind of technology for reducing with the time of monitoring fluorescence signal.Application
JP2009-142597 describes a kind of method for visualizing melanin, and this method is by multiphoton microscope inspection and FLIM
It is combined.In order to provide the good estimation of fluorescence lifetime, FLIM technologies need fluorescence signal over time and space integration,
It is expensive operation in terms of the time, so as to limit the possibility of application of this technology especially under three-dimensional imaging background.
The content of the invention
Accordingly, it would be desirable to the sensitive, method of Noninvasive be benefited from, for the three of the distribution of the melanin in biological tissue
Dimension visualization, this method is easy to implement, can obtain result that is reliable, quick, making a difference.
The purpose of the present invention is a kind of faster method simpler than art methods of development, and this method can be special
Melanin is detected to property, and its distributed in three dimensions in biological tissue, especially in epidermis can be characterized.
Also need to verify what is used during processing, for example based on anti-pigmentation activating agent, decolouring activating agent or
Promote effect or innocuousness of the product of pigmentation activating agent.
Also there is advantage in terms of the especially pigmentation of skin is characterized, to make melanin according to skin type, to plant
The quantity of group, geographical position or diet etc. and the difference of distribution embody, and so that the pigment for characterizing especially skin sinks
Disease, such as actinicity lenticula (actinic lentigenes), spot (freckle) or chloasma (go out sometimes in period of gestation
Existing spot).
In addition, in the case where seeking the background for rebuilding the new tissue of exploitation in the mode of synthesis, it is necessary to make melanin according to group
The quantity of the type of organization model, especially skin model and the difference of distribution embody.
Present invention aims at meet these all or part of needs.
Therefore, according to an aspect of the present invention, subject of the present invention is a kind of glimmering for the endogenous in biological tissue
Detection, quantization and/or the visualization method of light blob composition such as melanin, this method comprise the following steps：
A) after multiphoton excitation, gather what one group of continuous offer weakened on the fluorescence signal in sample with the time
The 3-D view or two dimensional image of information,
B) carry out linear regression to handle these images by the logarithm to the fluorescence signal, determine the linear regression
Slope spatial distribution in the sample, and
C) according at least to this three-dimensional spatial distribution, generate on fluorogen presence in the sample and/or amount
Information.
This method is non-invasive, can be used in non-treatment, in the case of beauty.
This method can assess pigmentation.
Acquired image is three-dimensional time image.Term " three-dimensional time image " is used to referring to a series of continuous with the time
3-D view.
Each 3-D view correspondence one iterates table in given time and the two dimensional image of the structure of the sample of given depth.
Each pixel of each 3-D view or two dimensional image can be limited by space coordinate (x, y, z), and z is in the sample
Depth.Therefore, a kind of special circumstances of each two dimensional image corresponding three-dimensional image, wherein, only adopted at given depth z
Collection.
The pixel of 3-D view has identical space coordinate by cluster together to the image gathered at each moment
In pixel set, each in these set is referred to as " time pixel ".
By being integrated in certain period of time to the fluorescence signal of two-photon excitation, the fluorescence letter of each pixel can be obtained
Number value.Especially, at regular intervals, (for example, equal to 1ns, 1.5ns, 2ns, 2.5ns especially between 1ns and 3ns
Or 3ns) period in, the integration of fluorescence signal can be carried out.
By the linear regression of the logarithm to fluorescence lifetime, the slope of fluorescence signal decrease can be obtained.This method can be needed
The three-dimensional time image of limited quantity is only gathered, for example, between three and five, this is carried between acquisition time and accuracy
Good trade off is supplied.The utilization of the slope obtained by the linear regression of logarithm promotes the limitation of the quantity of collection.
Compared with FLIM technologies, present invention reduces the quantity of time collection, this method is set to be more prone to and quicker
Implement on ground.Although FLIM technologies allow preferably to estimate fluorescence lifetime, as the reduction of the resolution ratio of 3-D view is, it is necessary to phase
When long acquisition time (about 30s), the method according to the invention is for obtaining three-dimensional visualization or quantifying to be more quick
And it is particularly advantageous, and the resolution ratio equivalent to microscopical theoretical resolution is kept simultaneously.
The acquisition time that the time pixel of coordinate (x, y, z) for giving sample is total is about 0.28 μ s, rather than
Several milliseconds (ms) in FLIM.
For example, the quantity for the time collection of each time pixel is equal to four, also, for each pixel, two-photon
The fluorescence signal excited particularly by 2ns regular intervals of time, and is integrated in the time between two collections.
The method according to the invention can be implemented by live body, can also be real in vitro (ex vivo) and in vitro sample
Apply this method.
The present invention improves the rapidity of acquisition method and image processing method.By the present invention, the 3D of tissue, which is characterized, is
Feasible, and allow high-throughout vivo applications.
Tissue may include human keratin material.Term " keratin material " is particularly for finger hair, eyelashes, eyebrow, skin
Skin, nail, mucous membrane, scalp.
According to the biological tissue of the present invention, especially keratin material can be natural or artificial；Sample is, for example,
Human skin or reconstruction or artificial skin.
Biological tissue can be the melanism cell of culture.
Information can with least one image, especially two dimension or 3-D view in the form of and generate.
Compared with other structural constituents, the information generated can provide in the tissue occupied by melanin
The information that the information of surface and/or volume and 3D on melanin are distributed.
According to another aspect of the present invention, subject of the present invention be it is a kind of assess particularly promote pigmentation, decolourize or
Anti- pigmented stimulation and/or the method for handling the effect to biological tissue, this method are following steps：
- pigmentation in biological tissue is assessed by method as discussed previously,
- sample is upset, the stimulation is selected from：Light radiation (especially solar radiation, ultraviolet (UVA (long wave ultraviolets
Line) and/or UVB (ultraviolet B radiation)) radiation or infrared ray (IR) radiation), cause stimulation and the mechanism of inflammatory reaction
(stretching, compacting, peeling, disengaging, stripping, abrasion) or make sample through being subject to processing, pigment is promoted especially with least one
Calm, decolouring resists pigmented product to make sample through being subject to processing,
- second of assessment is carried out to the pigmentation in biological tissue by method as discussed previously,
- compare the information generated during assessing twice, and
- this comparison is at least based on, assess the stimulation or handle to pigmented effect.
Processing can be the processing of non-treatment, especially beauty.
Processing can be selected from：Application, injection, intake or the suction of product, especially cosmetics.
Processing can be equivalent to taking food supplement and/or medicament.Processing can also include being selected biological tissue
From following processing：The application of product, especially cosmetics, injection, intake are sucked, or take food supplement and/or
Medicament.
Term " cosmetics " means that (it changes instruction 76/768/ according to the instruction 93/35/EEC on June 14th, 1993
EEC the product defined in).
The product can have decolorizing effect.Therefore, processing can include applying the pigmented chemistry examination of any regulation
Agent.
The method according to the invention can be used for assessing decolouring activating agent, anti-pigmentation activating agent or promote pigmentation
Effect or innocuousness of activating agent.Activating agent (for example, hydroquinones or retinoid) can be used for purpose and/or U.S. for the treatment of
The purpose of appearance.
The method according to the invention may be additionally used for some products (for example, skin cortex hormone of aadrenaline
(dermocorticoid) pigmented side effect) is estimated.
According to another aspect of the present invention, subject of the present invention is that a kind of assessment is stimulated and/or processing, especially anti-pigment are sunk
, promote pigmentation or the method decolourized to the effect of biological tissue, wherein, at least two regions of tissue differently by
Stimulate and/or be treated differently for printing, and wherein, compare before and after by the stimulation or the processing, by such as
Previously described method obtain on melanin presence in this region and/or the information of quantity.
For the effect of test product, the assessment done after the placebo treatment by product can be compared and led to
Cross the assessment done after this product treatment.
For example, placebo is the beauty medium as the beauty medium of the product for processing, but do not contain corresponding
Activating agent.
Can assess melanin in the individual biological tissue handled by beauty care compounds it is artificial or rebuild
Skin sample on distribution, and the same individual of the placebo treatments of beauty care compounds can passed through with melanin
Biological tissue in or the assessment of distribution on the identical sample of artificial or reconstruction skin compare.
Processing can correspond to apply product, especially cosmetics.For example, the product can have face cream, washing lotion, cream
Agent, oil, the form of powder, this inventory are unrestricted.
Product can be additionally included in carrier, to be applied in biological tissue, mankind's skin especially rebuild or artificial
On skin, the carrier is, for example, paster, dressing, bandage or facial mask.
Product can be not only to be used for sinking to the pigment that melanin quantity in biological tissues and/or distribution are worked
Product or decolouring product.Thus, for example, product can be used for beauty, for moisturizing or for protecting biological tissue, especially
Defence sunlight or for repairing biological tissue, and shadow is produced to melanin quantity in biological tissues and/or distribution simultaneously
Ring.Therefore, product can contain various compounds, especially except the activating agent that is worked for the melanin to biological tissue it
Outer activating agent.
This method can visualize melanin in biological tissues；For example, this method can learn melanin in skin
Distribution in each layer of the epidermis of skin.
According to another aspect of the present invention, another theme of the invention is a kind of for promotion processing, especially non-treatment
The method of processing, wherein, refer to the effect to melanin processing illustrated by method defined as above.
According to another aspect of the present invention, another theme of the invention is a kind of method comprised the following steps：In product
Selling period, for example, being referred in advertisement or in packaging confirms effect of product by method defined as above.
Brief description of the drawings
From the description of the non-limiting example for reading following executive mode of the invention and check the chart of accompanying drawing,
The present invention may be better understood, wherein：
Fig. 1 shows each step of the method according to the invention；
Fig. 2 is schematically and part shows the example of available multi-photon device in the method according to the invention；
Fig. 3 A to Fig. 3 D show the example of the IMAQ according to the present invention；
Fig. 4 A and Fig. 4 B show the various examples of the time change of the fluorescence in sample；
Fig. 5 shows that the parameter of the fluorescence lifetime for the section in constant depth dermatological specimens is represented；
Fig. 6 and Fig. 7 show the two-dimentional spot that the melanin obtained by the method according to the invention is distributed in dermatological specimens
Point and three-dimensional spots；
Fig. 8 shows the two-dimensional speckle being distributed by the melanin that the method for prior art is obtained in dermatological specimens；
The amount of the melanin in dermatological specimens before Fig. 9 A to Fig. 9 D show to handle and after processing；With
Figure 10 A, Figure 10 B and Figure 10 C show the human skin light type I according to the amount of melanin to human skin light type IV's
Characterize.
Embodiment
The step 1 of the method according to the invention can be by multiphoton microscope inspection, in biological organization sample
Each depth gathers multigroup two-dimensional time image.
For example using TCSPC (Single Photon Counting) photon counter, believed by the fluorescence of two-photon excitation
Integration number in certain period of time, can obtain each pixel.
The use of multiphoton microscope check device can allow being automatically brought into operation for biological tissue images acquisition step.
Especially, three-dimensional time image can be formed by a two-dimensional time image for being stacked in each depth.Often folding can be with
Including tens two-dimensional time images, it may for example comprise the image between 50 and 100, especially it is included in 65 and 80
Between image.
In step 2, for each pixel of coordinate (x, y, z), by calculating the logarithm of fluorescence intensity, to study fluorescence
The change of intensity over time.Then, by linear regression, the logarithm is adjusted, to estimate its slope, the slope and fluorescence longevity
Life is related.
Therefore, the image for the slope that the data obtained can be obtained by the linear regression of the logarithm by fluorescence intensity come
Represent.
The slope image of also referred to as parametric image contains two kinds of pixel：The pixel that is characterized with low slope and with height
The pixel that slope value is characterized.Pixel with high level slope is attributed to melanin, and other endogenous with the cell of epidermis are glimmering
Light blob (e.g., keratin, NAD (P) H, FAD (nucleic acid of flavin adenine two) etc.) is compared, and melanin has the shorter life-span.
In fact, melanin is characterized with two fluorescence lifetimes.Very short main fluorescence lifetime be about 0.1ns extremely
0.2ns (nanosecond).Another fluorescence lifetime of melanin is secondary and longer, between 0.7ns and 1.4ns.By than
Compared with other Intrinsic fluorescence groups (e.g., keratin, NAD (P) H, FAD etc.) of the cell of epidermis, which mainly have, is more than 1.5ns more
Long fluorescence lifetime.
Step 3 is to extract the pixel corresponding to melanin from slope image.For example, surface filtration is applied to each
Two dimensional image, the two dimensional image considers the size of melanosome, i.e. by the size of melanosome and about 1 μm2Reference surface face
Product compares, except denoising, more specifically to detect melanin.
For example, eliminating fluorescence signal with low value slope and with less than 1 μm2Size structure after,
Generate the information on melanin presence in the sample and/or amount.
The information can with the spatial distribution of melanin it is at least one represent in the form of, especially with one for sample
The form of the corresponding two dimensional image of the melanin spot of individual depth and generate.
Pair whole two dimensional image corresponding with given depth, can carry out the step 2 and the related spot of filtering of slope calculations
Step 3, also, for each image of 3-D stacks, repeat the step 2 and step 3.
This method can obtain the two-dimensional representation of a folded 3-D view for forming melanin spot.
, can be by calculating melanin density and/or black based on the three-dimensional melanin spot obtained by this method
Pigment spatial distribution characterizes melanin.
In view of the rapidity of calculating, this method can be used in screening technique.
It is any of to can be used to for measuring the multiphoton microscope inspection system that the system of fluorescence lifetime is combined
Implement above method.
Excitation wavelength used can be between 700nm and 1000nm, preferably about 760nm.This wave-length coverage can be with
Most of fluorescent components imaging to tissue.
Fig. 2 is schematically and part shows the example of available multi-photon device 100 in the method according to the invention.
For example, multi-photon device 100 is developed by Jenlab companiesType.
Device 100 includes femto-second laser 10, for example, titanium-sapphire (Ti：Sa) laser, the laser can be tuned
To infrared wavelength range, and the pulse of about 100 femtoseconds can be provided with about 80MHz repetition rate.Laser 10 is sent out
Go out infrared beam 11, the infrared beam 11 points to the laser beam scanning device 12 in the form of " XY scanners ".By scanning dress
The angular movement of 12 two galvanometer mirrors is put, the scanning of laser beam can be obtained.Then, light beam is reflected by dichronic mirror 14, then
It is focused on by object lens 15 on keratin material 13.Therefore, two galvanometer mirrors allow scanning in the propagation side with light beam
Focus point into (z-axis) vertical plane (x, y).
Piezo-electric device can translate object lens 15, so as to change the plane focused in keratin material 13.In this way,
Distributed in three dimensions of the melanin in keratin material can be rebuild by being superimposed acquired image.
Therefore, can be then detected in the signal produced by focal point, that is to say, that can be utilized by exciting object lens 15
Echo-planar imaging gathers (epicollection), can detect in the signal produced by focal point.First dichronic mirror 14 can be with
The formed multi-photon signal of selection, the especially autofluorescence (AF) from keratin material 13.
Then, the second dichronic mirror 16 can be by autofluorescence (AF) signal and for example corresponding to second_harmonic generation (SHG)
Other multi-photon Signal separators.Under any circumstance, signal passes through optical filter 17, reaches photon-counting detector (TCSPC：
(single photon counting of time correlation)) 18,19, so as to produce the image of combination, such as AF/SHG images.
First detector 18 and the second detector 19 can produce signal 18a and signal 19a, signal 18a and signal 19a
It is transferred into the electronic installation 20 for Signal sampling and processing.
Embodiment 1：The vivo observation of human skin
During testing, 3-D view one after the other is obtained to mankind's forearm live body, a 3-D view is by containing 70 two
The lamination composition of image is tieed up, the two dimensional image is in collected, the utilization from the surface from skin to 2.346 μm of depthDevice, is obtained by 760nm excitation wavelength and 40x/1.3NA object lens.
Fig. 3 A to Fig. 3 D show the time series of 3-D view, wherein, it illustrate only the skin in the forearm of healthy volunteer
On skin, close to depth of the basalis at 50 μm relative to skin surface of epidermis, four two-dimensional time images of live body collection.
For each temporal image, within 2ns period, fluorescence signal is integrated.
Under experimental conditions, for the point of the sample containing melanin, the most of fluorescent photons launched are at first
Between gather during be collected.The concentration of melanin is bigger, and the number of the photon of this first collection is more.According to intensity scale
Standard, Fig. 3 A arrow I corresponding to very first time passage represents the region of high melanin concentration.In this level, height
The melanin of concentration is formd with the fluorescence signal that higher intensity is rolled into a ball than other Intrinsic fluorescences.
Fig. 4 A and Fig. 4 B are shown for the pixel of the image corresponding with the point of the sample with melanin and with not having
There is the example of the time-evolution curve (Fig. 4 A) of the corresponding pixel of the point of the sample of melanin, and adjust glimmering by linear regression
The example of the logarithm (Fig. 4 B) of luminous intensity.
The two-dimensional representation of Fig. 5, Fig. 6 human skin in 50 μm of depth corresponding with Fig. 8.Fig. 5 is shown by fluorescence intensity
The parametric image for the slope that the linear regression of logarithm is obtained.The image has two kinds of pixel：It is characterized with low slope
Pixel and the pixel that is characterized with high slope value, the reasons why due to prior statement, the pixel that should be characterized with high slope value is returned
Because in melanin.
Fig. 6 is shown is attributed to the pixel of melanin by extracting, the two-dimensional speckle of the melanin obtained from Fig. 5, and
Fig. 8 shows the method by prior art, the result obtained according only to fluorescence intensity.
But when comparing Fig. 5 and Fig. 8, observe, because only highdensity pixel is attributed to melanin, therefore melanin is deposited
Underestimate by Fig. 8.
For each two dimensional image of 3-D stacks, corresponding to each pixel, according to the logarithm of the fluorescence intensity of time
The calculating of slope and the filtering of respective two-dimensional spot are repeated, to make the melanin spot of correlation dimensionally may be used as shown in Figure 7
Depending on change, and dimensionally to characterize melanin spot in whole epidermis, the distribution being included in cuticula.
Corresponding to the ensuing test of the embodiment 2 and embodiment 3 of progress by this method, melanin is illustrated
Quantity difference.
Embodiment 2：The assessment handled by skin cortex hormone of aadrenaline
Before and after being handled by skin cortex hormone of aadrenaline, to the same area of skin, such as previous institute is utilized
DescriptionMicroscope is observed.
Fig. 9 A show the original image of the fluorescence of the sample of skin area before treatment, and Fig. 9 C are shown by basis
The corresponding melanin spot that the method for the present invention is obtained.
(under occlusion) skin is handled by skin cortex hormone of aadrenaline.
Fig. 9 B and Fig. 9 D show the original image of the fluorescence of sample and after handling three weeks by according to the present invention's
The corresponding melanin spot that method is obtained.
The method according to the invention allows specific detection melanin；The comparative descriptions of spot shown in Fig. 9 C and Fig. 9 D
The quantity of melanin is reduced, so as to allow to quantify by the three-dimensional of melanin in skin, is assessed more well by skin
Change caused by cortex hormone of aadrenaline.
Embodiment 3：Human skin light type I to human skin light type IV sign
Human skin is classified into six kinds of light type according to the reaction that it is exposed under the sun.Dark skin (light type V and light
Type VI) there is greater number of melanin, the melanin shields ultraviolet (UV) line naturally.The method according to the invention allows more
Analyze well corresponding to scope from very pale to the skin of medium brown light type I to light type IV composition pigmentation.
Figure 10 A and Figure 10 B show the original image of the fluorescence of the dermatological specimens for various light type and by according to this
The corresponding melanin spot that the method for invention is obtained.
According to Figure 10 B spot, the 3D density of melanin is calculated, to obtain Figure 10 C comparison figure, this, which compares figure, proves
The quantity of melanin increases with light type.
As shown in the spot by Figure 10 B corresponding light type I, the method according to the invention can quantify depositing for melanin
Even if melanin is under low concentration.Therefore, this method can be used for any kind of tissue containing melanin.
Claims (15)
1. a kind of detection, quantization and/or the visualization method of the Intrinsic fluorescence group being used in biological tissue, methods described bag
Include following steps：
A) after multiphoton excitation, one group of continuous 3-D view or two dimensional image, the 3-D view or X-Y scheme are gathered
As providing the information weakened on the fluorescence signal in sample with the time；
B) this is handled by calculating the logarithm of the fluorescence signal and carrying out linear regression to the logarithm of the fluorescence signal
A little images, determine the spatial distribution of the slope of the linear regression in the sample；With
C) spatial distribution is at least based on, is generated on fluorogen presence in the sample and/or bulk density or surface
The information of density,
The fluorogen is melanin.
2. according to the method described in claim 1, wherein, one group of image is included between three images and five images.
3. method according to claim 1 or 2, wherein, the fluorescence signal is integrated at regular intervals.
4. method according to claim 3, wherein, the fluorescence signal is accumulated in the period between 1ns and 3ns
Point.
5. according to the method described in claim 1, wherein, after the pixel with low value slope has been eliminated, generation step c)
Described information.
6. according to the method described in claim 1, wherein, step c) described information with the spatial distribution of the melanin extremely
Lack a kind of form of expression and generate.
7. according to the method described in claim 1, wherein, the information that is generated provide in the tissue by melanin institute
The surface occupied and/or the information of volume.
8. according to the method described in claim 1, wherein, methods described is implemented to biological tissue live body.
9. according to the method described in claim 1, wherein, it is described be organized as human skin, rebuild artificial skin or training
Foster melanism cell.
10. a kind of be used to assess the method for stimulating and/or handling the effect to biological tissue, it the described method comprises the following steps：
- pigmentation in the tissue is assessed by the method according to any one of claim 1-9,
- by the method according to any one of claim 1-9 to the tissue for the effect for being upset or having handled
In pigmentation carry out second and assess, the stimulation is selected from：Light radiation；Cause the stimulation of inflammatory reaction；And mechanism,
- compare the information generated during assessing twice, and
- at least compared based on this, the stimulation or the processing are assessed to pigmented effect.
11. method according to claim 10, wherein, the processing is to promote pigmentation or decolouring.
12. method according to claim 10, wherein, the light radiation is solar radiation, ultraviolet UV radiation or infrared
Line IR is radiated.
13. method according to claim 10, wherein, the processing is pigmented or de- using at least one promotion
The processing of the cosmetics of color.
14. a kind of be used to assess the method for stimulating and/or handling the effect to biological tissue, wherein, at least the two of the tissue
Individual region is differently upset and/or is treated differently for printing, and wherein, compares by the stimulation or the processing
It is preceding and afterwards, by the method according to any one of claim 1 to 8 obtain on melanin in this region
Presence and/or amount information.
15. method according to claim 14, wherein, the processing is anti-pigmentation, promotes pigmentation or decolouring.
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| FR1160172A FR2982369B1 (en) | 2011-11-08 | 2011-11-08 | NON-INVASIVE METHOD FOR SPECIFIC 3D DETECTION, VISUALIZATION AND / OR QUANTIFICATION OF A ENDOGENEOUS FLUOROPHORE SUCH AS MELANIN WITHIN A BIOLOGICAL TISSUE |
| FR1160172 | 2011-11-08 | ||
| PCT/IB2012/056230 WO2013068943A1 (en) | 2011-11-08 | 2012-11-07 | Non-invasive method for specific 3d detection, visualization and/or quantification of an endogenous fluorophore such as melanin in a biological tissue |
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| CN104297225B (en) * | 2014-09-29 | 2018-02-16 | 无限极（中国）有限公司 | A kind of quick detection of intracellular melanin and the method for imaging |
| CN105852808B (en) * | 2015-05-08 | 2019-08-30 | 江苏坤辉生物科技有限公司 | A kind of method and its detection device of the UV light-induced skin injury of living body Non-invasive detection |
| JPWO2018008136A1 (en) | 2016-07-07 | 2019-04-18 | オリンパス株式会社 | Image processing apparatus and operation method of image processing apparatus |
| US10732100B2 (en) * | 2018-06-29 | 2020-08-04 | L'oreal | Systems and methods for predicting sun protection factor of sunscreen formulations in vitro |
| US11333607B2 (en) | 2018-10-02 | 2022-05-17 | Electronics And Telecommunications Research Institute | Fluorescent signal detection apparatus using diagnostic kit |
| CN114246548B (en) * | 2020-09-23 | 2024-05-24 | 中国中医科学院医学实验中心 | Device and system for detecting acne inflammation and application thereof |
| WO2024060018A1 (en) * | 2022-09-20 | 2024-03-28 | Juan Liu | Non-invasive method for detection, visualization and/or quantification of an endogenous fluorophore such as melanin in a biological tissue |
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