CN103930767A - Non-invasive method for specific 3d detection, visualization and/or quantification of an endogenous fluorophore such as melanin in a biological tissue - Google Patents
Non-invasive method for specific 3d detection, visualization and/or quantification of an endogenous fluorophore such as melanin in a biological tissue Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
- A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
- A61B5/443—Evaluating skin constituents, e.g. elastin, melanin, water
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/48—Other medical applications
- A61B5/4848—Monitoring or testing the effects of treatment, e.g. of medication
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
- G06T7/0014—Biomedical image inspection using an image reference approach
- G06T7/0016—Biomedical image inspection using an image reference approach involving temporal comparison
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/90—Determination of colour characteristics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
- A61B5/7235—Details of waveform analysis
- A61B5/7242—Details of waveform analysis using integration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N2021/178—Methods for obtaining spatial resolution of the property being measured
- G01N2021/1785—Three dimensional
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10024—Color image
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10056—Microscopic image
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- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10064—Fluorescence image
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
- G06T2207/30088—Skin; Dermal
Abstract
Method for detection, quantification and/or visualization of an endogenous fluorophore, in a biological tissue, the method comprising the steps consisting in: a) acquiring, after multiphoton excitation, a set of successive three- dimensional or two-dimensional images providing information on the decrease over time in the fluorescence signals in the sample, b) determining by processing these images, by performing a linear regression of the logarithm of the fluorescence signals, the spatial distribution in the sample of the slopes of said linear regression, and c) generating information on the presence and/or the volume density or surface density of a fluorophore in the sample at least on the basis of this spatial distribution, the fluorophore being melanin.
Description
Technical field
The present invention relates to biological tissue, especially such as the observation of the keratin material of skin.
The present invention more specifically but not only relate to the melanic distribution that is intended to determine in biological tissue, quantity and/or the character especially distributing, the effect of especially processing in order to assess cosmetic treatments or treatment.
Background technology
The color of biological tissue and melanic quantity and distributed in three dimensions have relation closely.Nowadays, with two dimensional form, by utilizing the melanic white light imaging of Fontana Masson dyeing with quantitative in Histological section, or with three dimensional form, after image is processed, by melanic multi-photon imaging and 3-D quantitative, can characterize melanic quantity and distribution.
Multiphoton microscope inspection promotes biological tissue, the especially deep observation of the tissue of the work of height scattering.This is because because infrared light is by less scattering and by less absorption, therefore infrared light penetrates in tissue better, and utilizes Nonlinear Mechanism to produce signal and in scattering medium, has introduced stronger signal choice criteria.This Nonlinear Mechanism interacts when relating to molecule that two kinds or even more kinds of photon and expectation detect or structure.Conventionally, excitation beam is focused and scans in sample, detects formed nonlinear properties, to obtain image.Owing to having the non-limiting dependence of the signal of exciting power, the singularity of such imaging is the optical section performance based on provided.Because exciting power density is very high at focus place, therefore the nonlinear effect of considering only appears in limited volume of focus effectively; This key property has been guaranteed the control of the signal that obtains in sample, therefore can obtain the intrinsic dimensional resolution of about micron.Imaging depth changes according to tissue, and for human skin, imaging depth can be approximately 130 μ m to 200 μ m.
In addition,, owing to using endogenous signal source, this imaging technique does not need tagged tissue composition.By endogenous chromophore, form fluorescence signal; Except melanin, can mention NAD (P) H (nicotinamide-adenine dinucleotide phosphate), flavine, keratin and elastin laminin.
Application FR 2 944 425 discloses a kind of by the method for multiphoton microscope inspection and evaluation cutaneous pigmentation, the method, based in skin, in the level of the basalis of epidermis, reflects the melanin of high concentration by the stronger fluorescence intensity of Intrinsic fluorescence group than other.There is high-intensity pixel owing to melanin.Yet, this method is not always satisfactory, this be because, first, the method can by other the fluorophore that also demonstrates high fluorescent (as, keratin) upset, secondly, the method reckons without the melanin with the fluorescence signal that can mention in the same breath with other Intrinsic fluorescence groups.
More specific method comprises considers melanic fluorescence lifetime.Really, melanic fluorescence lifetime depends on melanic primary fluorescence character, does not depend on fluorescence intensity and the fluorophore concentration in excitation volume.Melanin has the two-photon fluorescence excitation life-span, and this fluorescence lifetime is different from the fluorescence lifetime of other Intrinsic fluorescence groups.According to the image obtaining by FLIM (fluorescence lifetime imaging), can estimate this fluorescence lifetime, this FLIM is article " Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy " (the European Journal of Pharmaceutics and Biopharmaceutics people such as M.S.Roberts, the 77th volume in 2011, the 469th page to the 488th page) described in, comprise a kind of technology that monitoring fluorescence signal reduces in time.Application JP2009-142597 has described a kind of for visual melanic method, and the method combines multiphoton microscope inspection and FLIM.For the good estimation of fluorescence lifetime is provided, the integration of the fluorescence signal of FLIM Technology Need on time and space, aspect the time, be expensive operation, thereby limiting the especially possibility of the application under three-dimensional imaging background of this technology.
Summary of the invention
Therefore, need to benefit from method sensitive, Noninvasive, for the three-dimensional visualization of the melanic distribution in biological tissue, the method is easy to implement, can obtain reliably, fast, the result of making a difference.
The object of the invention is to develop that a kind of the method can detect melanin specifically than art methods method more simply faster, and can characterize Qi biological tissue, the distributed in three dimensions in epidermis especially.
Also need to verify during processing and to use, for example, based on anti-pigmentation activating agent, decolouring activating agent or promote effect or the innocuousness of the product of pigmentation activating agent.
Also there is advantage characterizing aspect the pigmentation of skin especially, so that making melanin specializes according to the difference of the quantity of skin type, population, geographic position or diet etc. and distribution, and to characterize the especially pigmentation disease of skin, as actinicity lenticula (actinic lentigenes), spot (freckle) or chloasma (spot sometimes occurring at period of gestation).
In addition, seeking under the background of rebuilding the new tissue of exploitation in synthetic mode, need to make melanin specialize according to the difference of the quantity of the type of organize models, especially skin model and distribution.
The object of the invention is to meet these needs of all or part.
Therefore, according to an aspect of the present invention, theme of the present invention be a kind of Intrinsic fluorescence group composition in biological tissue as melanic detection, quantification and/or visualization method, the method comprises the following steps:
A) after multiphoton excitation, gather the one group of continuous 3-D view that the information weakening in time about the fluorescence signal in sample is provided or two dimensional image,
B) by being carried out to linear regression, the logarithm of described fluorescence signal processes these images, the space distribution of the slope of determining described linear regression in described sample, and
C), at least according to this three-dimensional spatial distribution, generate the information of about the fluorophore existence in described sample and/or amount.
The method right and wrong are invasive, can be used in situation non-treatment, beauty treatment.
The method can be assessed pigmentation.
The image gathering is three-dimensional time image.Term " three-dimensional time image " is used in reference to a series of continuous 3-D view in time.
Each 3-D view correspondence one iterates table at the two dimensional image of the structure of the sample of given time and given depth.
Each pixel of each 3-D view or two dimensional image can be limited by volume coordinate (x, y, z), and z is the degree of depth in sample.Therefore, a kind of special circumstances of each two dimensional image corresponding three-dimensional image, wherein, only gather at given depth z place.
The pixel of 3-D view is trooped together in the pixel set of the identical volume coordinate of having of the image constantly gathering at each, and each in these set is called as " time pixel ".
By the fluorescence signal integration to two-photon excitation in certain hour section, can obtain the value of the fluorescence signal of each pixel.Especially, with the interval of rule, for example, in the time period that especially (, equals 1ns, 1.5ns, 2ns, 2.5ns or 3ns) between 1ns and 3ns, can carry out the integration of fluorescence signal.
By the linear regression to the logarithm of fluorescence lifetime, can obtain the slope that fluorescence signal weakens.The method can only gather the three-dimensional time image of limited quantity, and for example, between three and five, this provides good trading off between acquisition time and degree of accuracy.The utilization of the slope that the linear regression by logarithm obtains has promoted this restriction of the quantity that gathers.
Compare with FLIM technology, the present invention has reduced the quantity of time collection, the method is more prone to and implements more rapidly.Although FLIM technology allows to estimate better fluorescence lifetime, but the reduction along with the resolution of 3-D view, acquisition time that need to be quite long (approximately 30s), the method according to this invention is more fast and particularly advantageous for obtaining three-dimensional visualization or quantification, and keeps being equivalent to the resolution of microscopical theoretical resolution simultaneously.
The total acquisition time of time pixel for the coordinate (x, y, z) of given sample is approximately 0.28 μ s, rather than several milliseconds (ms) in FLIM.
For example, the quantity gathering for time of each time pixel equals four, and for each pixel, the fluorescence signal of two-photon excitation, in the time between two collections, especially by the regular intervals of time of 2ns, and is integrated.
The method according to this invention can be implemented by live body, also can implement the method to sample (ex vivo) and in vitro in vitro.
The present invention has improved the rapidity of acquisition method and image processing method.Rely on the present invention, it is feasible that the 3D of tissue characterizes, and allows high-throughout vivo applications.
Tissue can comprise human keratin material.Term " keratin material " is particularly useful for that finger is sent out, eyelashes, eyebrow, skin, nail, mucous membrane, scalp.
According to biological tissue of the present invention, especially keratin material can be natural or artificial; Sample is for example skin human skin or reconstruction or artificial.
Biological tissue can be the melanism cell of cultivating.
Information can generate with the form of at least one image, especially two dimension or 3-D view.
Compare with other structural constituents, the information generating can provide about in described tissue by the information of the occupied surface of melanin and/or volume and the information that distributes about melanic 3D.
According to a further aspect in the invention, theme of the present invention is that a kind of assessment particularly promotes pigmentation, decolouring or anti-Pigmented stimulation and/or processes the method to the effect of biological tissue, and the method is following steps:
-by the pigmentation in method assessment biological tissue as discussed previously,
-sample is upset, this stimulation is selected from: stimulation and the mechanical effect (stretching, compacting, peeling, depart from, peel off, wear and tear) of optical radiation (especially solar radiation, ultraviolet ray (UVA (long wave ultraviolet) and/or UVB (ultraviolet B radiation)) radiation or infrared ray (IR) radiation), the reaction that causes inflammation or make sample through being subject to processing, especially utilize at least one to promote pigmentation, decolouring or anti-Pigmented product to make sample through being subject to processing
-by method as discussed previously, pigmentation in biological tissue is assessed for the second time,
The information that-comparison generates during twice assessment, and
-at least based on this comparison, assess described stimulation or process Pigmented effect.
Processing can be processing non-treatment, that especially improve looks.
Processing can be selected from: the applying, inject, take in or suck of product, especially cosmetics.
Processing can be equivalent to take food supplement and/or medicament.Process and also can comprise and makes biological tissue be subject to being selected from following processing: the applying, inject, take in or suck of product, especially cosmetics, or take food supplement and/or medicament.
Term " cosmetics " means according to the product defined in the instruction 93/35/EEC on June 14th, 1993 (its modify instruction 76/768/EEC).
This product can have decolorizing effect.Therefore, processing can comprise and uses the Pigmented chemical reagent of any adjusting.
The method according to this invention can be used to assessment decolouring activating agent, anti-pigmentation activating agent or promote effect or the innocuousness of pigmentation activating agent.Activating agent (for example, p-dihydroxy-benzene or retinoid) object that can be used for the treatment of and/or the object of beauty treatment.
The method according to this invention also can be used to the Pigmented spinoff of some product (for example, skin cortex hormone of aadrenaline (dermocorticoid)) to assess.
According to a further aspect in the invention, theme of the present invention is that a kind of assessment stimulates and/or processing, especially anti-pigmentation, promotion pigmentation or the method for decolouring to the effect of biological tissue, wherein, at least two regions of tissue are differently upset and/or are differently processed, and wherein, relatively before being subject to described stimulation or described processing and afterwards, the existence in described region about melanin obtaining by method as discussed previously and/or the information of quantity.
For the effect of test products, the assessment that can relatively do after the placebo treatment by product and the assessment of doing after by this product treatment.
For example, placebo be with for the treatment of the same beauty treatment medium of the beauty treatment medium of product, but do not contain corresponding activating agent.
Can assess melanin in by the individual biological tissue of beauty treatment compound treatment or the sample of artificial or the skin rebuild on distribution, and can compare with the assessment of the distribution of melanin in the biological tissue of the same individuality of the placebo treatment by beauty treatment compound or on the same sample of skin artificial or that rebuild.
Processing can be corresponding to applying product, especially cosmetics.For example, this product can have the form of face cream, washing lotion, paste, oil, powder, and this inventory is unrestricted.
Product also can be included in carrier, and to be applied in biological tissue, on especially that rebuild or artificial human skin, this carrier is for example paster, dressing, bandage or facial mask.
Product can be not only pigmentation product or the decolouring product working for the quantity in biological tissue and/or distribution to melanin.Therefore, for example, product can be used to beauty treatment, for moisturizing or for the protection of biological tissue, especially defend sunlight or for repairing biological tissue, and the quantity in biological tissue and/or distribution exerts an influence to melanin simultaneously.Therefore, product can contain various compounds, especially the activating agent except the activating agent that is used for the melanin of biological tissue to work.
The melanin of the method in can Shi biological tissue is visual; For example, the method can be learnt the distribution of melanin in each layer of the epidermis of skin.
According to a further aspect in the invention, another theme of the present invention is a kind of for promoting to process, being especially the method that non-treatment is processed, and wherein, mentions by as the effect that melanin is processed that the method that above limits illustrates.
According to a further aspect in the invention, another theme of the present invention is a kind of method comprising the steps: at the selling period of product, for example, in advertisement or mention on the packaging by as the method that above limits confirms the effect of product.
Accompanying drawing explanation
The description of the non-limiting example of the executive mode of the present invention below reading and the chart of checking accompanying drawing, the present invention may be better understood, wherein:
Fig. 1 illustrates each step of the method according to this invention;
Fig. 2 schematically and the local example that available multi-photon device is in the method according to the invention shown;
Fig. 3 A to Fig. 3 D illustrates the example according to image acquisition of the present invention;
Fig. 4 A and Fig. 4 B are illustrated in the various examples of the time variation of the fluorescence in sample;
Fig. 5 illustrates the Parametric Representation for the fluorescence lifetime of the section at constant depth dermatological specimens;
Fig. 6 and Fig. 7 illustrate two-dimensional speckle and the three-dimensional spot that the melanin that obtains by the method according to this invention distributes in dermatological specimens;
Fig. 8 illustrates the two-dimensional speckle that melanin that the method by prior art obtains distributes in dermatological specimens;
Melanic amount in dermatological specimens before Fig. 9 A to Fig. 9 D illustrates and processes and after processing; With
Figure 10 A, Figure 10 B and Figure 10 C illustrate the sign to human skin light type IV according to the human skin light type I of melanic amount.
Embodiment
The step 1 of the method according to this invention can be to check by multiphoton microscope, in each degree of depth collection of biological organization sample, organizes two-dimensional time image more.
For example utilize TCSPC (Single Photon Counting) photon counter, the integration of the fluorescence signal by two-photon excitation in certain hour section, can obtain each pixel.
The use of multiphoton microscope testing fixture can allow the automatic operation of biological tissue images acquisition step.
Especially, three-dimensional time image can be stacked in the two-dimensional time image of each degree of depth and form by one.Often fold and can comprise tens two-dimensional time images, for example, be included in the image between 50 and 100, be especially included in the image between 65 and 80.
In step 2, for each pixel of coordinate (x, y, z), by calculating the logarithm of fluorescence intensity, study the variation of fluorescence intensity along with the time.Subsequently, by linear regression, adjust this logarithm, to estimate its slope, this slope is relevant to fluorescence lifetime.
The image of the slope that the data that therefore, obtain can obtain by the linear regression of the logarithm by fluorescence intensity represents.
Slope replot also referred to as parametric image looks like the pixel that contains two types: the pixel that the pixel that the low slope of take is feature and the high slope value of take are feature.There is the pixel of high value slope owing to melanin, with other Intrinsic fluorescence groups of the cell of epidermis (as, keratin, NAD (P) H, FAD (flavin adenine two nucleic acids) etc.) compare, melanin has the shorter life-span.
In fact, to take two fluorescence lifetimes be feature to melanin.Very short main fluorescence lifetime is approximately 0.1ns to 0.2ns (nanosecond).Melanic another fluorescence lifetime is less important and long, between 0.7ns and 1.4ns.By relatively, other Intrinsic fluorescence groups of the cell of epidermis (as, keratin, NAD (P) H, FAD etc.) mainly there is the longer fluorescence lifetime that is greater than 1.5ns.
Step 3 is to extract corresponding to melanic pixel from slope replot picture.For example, surface filtration is applied to each two dimensional image, this two dimensional image has been considered the size of melanosome, that is, and and by the size of melanosome and about 1 μ m
2reference surface Area comparison so that except denoising, more specifically detect melanin.
For example, there is the fluorescence signal of low value slope and have and be less than 1 μ m having eliminated
2the structure of size after, generated about melanin existence in sample and/or the information of amount.
This information can be with the form of at least one expression of melanic space distribution, with the form of the corresponding two dimensional image of the melanin spot of a degree of depth with for sample, generate especially.
To the whole two dimensional image corresponding with given depth, can carry out the step 2 and the step 3 of filtering relevant spot of slope calculations, and each image for 3-D stacks, can repeat this step 2 and step 3.
The method can obtain a folded two-dimensional representation that forms the 3-D view of melanin spot.
Three-dimensional melanin spot based on obtaining by this method, can characterize melanin by calculating melanin density and/or melanin space distribution.
Consider the rapidity of calculating, this method can be used in screening technique.
Any known with for measuring the multiphoton microscope check system that the system of fluorescence lifetime combines, can be used to implement method above.
Excitation wavelength used can, between 700nm and 1000nm, be preferably approximately 760nm.This wavelength coverage can be to most of fluorescent component imagings of organizing.
Fig. 2 schematically and the local example that available multi-photon device 100 is in the method according to the invention shown.
For example, multi-photon device 100Wei You Jenlab company develops
type.
Device 100 comprises femto-second laser 10, for example, titanium-sapphire (Ti:Sa) laser instrument, this laser instrument can be tuned to infrared wavelength range, and the pulse of about 100 femtoseconds can be provided with the repetition rate of about 80MHz.Laser instrument 10 sends infrared beam 11, the laser beam flying device 12 that this infrared beam 11 points to the form of " XY scanner ".The angular motion of two galvanometer mirrors by scanister 12, can obtain the scanning of laser beam.Then, light beam is reflected by dichronic mirror 14, then by object lens 15, is focused on keratin material 13.Therefore, two galvanometer mirrors allow the focus point of scanning in the direction of propagation with light beam (z axle) vertical plane (x, y).
Piezo-electric device can make object lens 15 translations, thereby changes the plane focusing in keratin material 13.In this way, the image that can gather by stack, rebuilds the distributed in three dimensions of melanin in keratin material.
, so the signal producing in focus place can be detected, that is to say for this reason, can utilize echo-planar imaging collection (epicollection) by exciting object lens 15, can detect the signal producing in focus place.The first dichronic mirror 14 can be selected formed multi-photon signal, especially derives from the autofluorescence (AF) of keratin material 13.
Then, the second dichronic mirror 16 can be by autofluorescence (AF) signal with for example separated corresponding to other multi-photon signals of second harmonic generation (SHG).Under any circumstance, signal through optical filter 17, arrives photon-counting detector (single photon counting of TCSPC:(time correlation)) 18,19, thus can produce the image of combination, AF/SHG image for example.
The first detecting device 18 and the second detecting device 19 can produce signal 18a and signal 19a, and this signal 18a and signal 19a are transferred into the electronic installation 20 for signals collecting and processing.
embodiment 1: the vivo observation of human skin
At test period, one after the other mankind's forearm live body is obtained to 3-D view, a 3-D view is comprised of the lamination that contains 70 two dimensional images, and this two dimensional image is collected to the degree of depth of 2.346 μ m on the surface from from skin, utilizes
device, obtains by the excitation wavelength of 760nm and the object lens of 40x/1.3NA.
Fig. 3 A to Fig. 3 D illustrates the time series of 3-D view, wherein, only show on the skin of healthy volunteer's forearm, near the basalis of epidermis at the depth with respect to skin surface 50 μ m, four two-dimensional time images that live body gathers.For each temporal image, within the period of 2ns, fluorescence signal is carried out to integration.
Under experiment condition, for the point that contains melanic sample, most of fluorescent photons of launching are collected during the very first time gathers.Melanic concentration is larger, and the number of this first photon gathering is just more.According to strength criterion, Fig. 3 A represents the region of high melanin concentration corresponding to the arrow I of very first time passage.In this level, the melanin of high concentration has formed the fluorescence signal with the intensity higher than other Intrinsic fluorescence groups.
Fig. 4 A and Fig. 4 B show for the pixel of the corresponding image of the point with thering is melanic sample and with the example of the time-evolution curve (Fig. 4 A) of the corresponding pixel of the point without melanic sample, and by linear regression, adjust the example of the logarithm (Fig. 4 B) of fluorescence intensity.
Fig. 5, Fig. 6 and Fig. 8 correspondence are in the two-dimensional representation of the human skin of the 50 μ m degree of depth.Fig. 5 illustrates the parametric image of the slope that the linear regression by the logarithm of fluorescence intensity obtains.This image has the pixel of two types: the pixel that the pixel that the low slope of take is feature and the high slope value of take are feature, due to the reason of prior statement, the pixel that the high slope value of should take is feature is owing to melanin.
Fig. 6 shows by extracting owing to melanic pixel, the melanic two-dimensional speckle obtaining from Fig. 5, and Fig. 8 shows the method by prior art, the result only obtaining according to fluorescence intensity.
But when comparison diagram 5 and Fig. 8, observe, because highdensity pixel is only owing to melanin, therefore melanic existence is underestimated by Fig. 8.
Each two dimensional image for 3-D stacks, corresponding to each pixel, according to the filtration of the calculating of the slope of the logarithm of the fluorescence intensity of time and respective two-dimensional spot, be repeated, to make as shown in Figure 7 relevant melanin spot dimensionally visual, and so that dimensionally characterize melanin spot at whole epidermis, be included in the distribution in cuticula.
Corresponding to the test of embodiment 2 by this method ensuing and that carry out and embodiment 3, the difference of melanic quantity has been described.
embodiment 2: the assessment of processing by skin cortex hormone of aadrenaline
Before processing by skin cortex hormone of aadrenaline and afterwards, the same area to skin, utilizes as described earlier
microscope is observed.
Fig. 9 A shows the original image of the fluorescence of the sample of skin area before processing, and Fig. 9 C shows the corresponding melanin spot obtaining by the method according to this invention.
(under circumstance of occlusion) processes skin by skin cortex hormone of aadrenaline.
Fig. 9 B and Fig. 9 D show the original image of fluorescence of sample and the corresponding melanin spot obtaining by the method according to this invention after processing three weeks.
The method according to this invention allows specific detection melanin; The melanic quantity of comparative descriptions of the spot shown in Fig. 9 C and Fig. 9 D reduces, thereby allows, by melanic three-dimensional quantification the in skin, to assess more well by the caused change of skin cortex hormone of aadrenaline.
embodiment 3: human skin light type I is to the sign of human skin light type IV
The reaction that human skin is exposed under the sun according to it is classified into six kinds of light types.Dark skin (light type V and light type VI) has the melanin of greater number, and this melanin shields ultraviolet (UV) line naturally.The method according to this invention allows to analyze better the light type I of the skin from very pale to medium brown corresponding to scope to the pigmentation of the formation of light type IV.
Figure 10 A and Figure 10 B show for the original image of the fluorescence of the dermatological specimens of various smooth types and the corresponding melanin spot that obtains by the method according to this invention.
According to the spot of Figure 10 B, calculate melanic 3D density, to obtain the comparison diagram of Figure 10 C, this comparison diagram has proved that melanic quantity increases with light type.
Shown in the spot of the corresponding light type I by Figure 10 B, the method according to this invention can quantize melanic existence, even if melanin is under low concentration.Therefore, the method can be used for melanic tissue of containing of any type.
Claims (10)
1. detection, quantification and/or the visualization method for the Intrinsic fluorescence of biological tissue, rolled into a ball, said method comprising the steps of:
A) after multiphoton excitation, gather one group of continuous 3-D view or two dimensional image, described 3-D view or two dimensional image provide the information weakening in time about the fluorescence signal in sample;
B) by being carried out to linear regression, the logarithm of described fluorescence signal processes these images, the space distribution of the slope of determining described linear regression in described sample; With
C) at least based on this space distribution, generate the information of about the fluorophore existence in described sample and/or volume density or superficial density,
Described fluorophore is melanin.
2. method according to claim 1, wherein, described one group of image comprises three image to five images.
3. method according to claim 1 and 2, wherein, with the interval of rule, especially, in the period between 1ns and 3ns, carries out integration to described fluorescence signal.
4. according to the method described in any one in aforementioned claim, wherein, after eliminating the pixel with low value slope, generate described information.
5. according to the method described in any one in aforementioned claim, wherein, described information generates with the form of at least one expression of described melanic space distribution.
6. according to the method described in any one in aforementioned claim, wherein, the information generating provide about in described tissue by the information of the occupied surface of melanin and/or volume.
7. according to the method described in any one in aforementioned claim, wherein, biological tissue's live body is implemented to described method.
8. according to the method described in any one in aforementioned claim, wherein, described in the melanism cell that is organized as skin human skin, reconstruction or artificial or cultivates.
9. for assessment of stimulating and/or processing, especially promote pigmentation or the method for decolouring to the effect of biological tissue, said method comprising the steps of:
-by assessing the pigmentation in described tissue according to the method described in any one in aforementioned claim,
-by the pigmentation in the described tissue of the effect that is upset or processes being assessed for the second time according to the method described in any one in aforementioned claim, described stimulation is selected from: optical radiation, especially solar radiation, ultraviolet (UV) radiation or infrared ray IR radiation; The stimulation that causes inflammation and react; And mechanical effect, described processing especially utilizes at least one processing that promotes the cosmetics of Pigmented or decolouring, and described promotion cosmetics Pigmented or decolouring are for example lucinol or the Pigmented chemical reagent of any other adjusting,
The information that-comparison generates during twice assessment, and
-at least based on this relatively, assess described stimulation or described processing to Pigmented effect.
10. one kind for assessment of stimulating and/or processings, especially anti-pigmentation, promote pigmentation or the method for decolouring to the effect of biological tissue, wherein, at least two regions of described tissue are differently upset and/or are differently processed, and wherein, relatively before being subject to described stimulation or described processing and afterwards, by the existence in described region about melanin that obtains according to the method described in any one in claim 1 to 9 and/or the information of amount.
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297225A (en) * | 2014-09-29 | 2015-01-21 | 无限极（中国）有限公司 | Method for quickly detecting and imaging melanin in cells |
| WO2016180095A1 (en) * | 2015-05-08 | 2016-11-17 | 上海交通大学 | Method for detecting, in noninvasive in-vivo manner, skin damage induced by ultraviolet light, and detection device 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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| FR3024226B1 (en) * | 2014-07-23 | 2019-05-10 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | METHOD AND SYSTEM FOR DETECTING FROST |
| WO2018008136A1 (en) * | 2016-07-07 | 2018-01-11 | オリンパス株式会社 | Image processing device and operation method for image processing device |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2944425A1 (en) * | 2009-04-20 | 2010-10-22 | Oreal | Stimulus or cosmetic treatment pigmentation or action evaluating method for distribution of melanin in lucinol on three-dimensional human skin model, involves generating information representing action of stimulus/treatment based on result |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919710A (en) * | 1996-07-18 | 1999-07-06 | The United States Of America As Represented By The Secretary Of The Air Force | Optical method for quantitating dissolved oxygen in fuel |
| JP2003329588A (en) * | 2002-05-14 | 2003-11-19 | Masaya Fukui | Method, system, and apparatus for identifying material |
| US9750449B2 (en) * | 2003-12-12 | 2017-09-05 | Johnson & Johnson Consumer Inc. | Method of assessing skin |
| JP2005274308A (en) * | 2004-03-24 | 2005-10-06 | Shiseido Co Ltd | Method for screening long-wavelength ultraviolet ray induction melanization inhibitor, and method for evaluating long-wavelength ultraviolet induction melanization suppression effect |
| JP5147379B2 (en) * | 2007-12-18 | 2013-02-20 | 花王株式会社 | Method and apparatus for visualizing melanin distribution |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2944425A1 (en) * | 2009-04-20 | 2010-10-22 | Oreal | Stimulus or cosmetic treatment pigmentation or action evaluating method for distribution of melanin in lucinol on three-dimensional human skin model, involves generating information representing action of stimulus/treatment based on result |
Non-Patent Citations (4)
| Title |
|---|
| ANA-MARIA PENA ET.AL: "Multiphoton microscopy of engineering dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast", 《JOURNAL OF BIOMEDICAL OPTICS》, vol. 15, no. 5, 29 October 2010 (2010-10-29), XP055017519, DOI: doi:10.1117/1.3503411 * |
| ANTHONY M.D.LEE ET.AL: "In vivo video rate multiphoton microscopy imaging of human skin", 《OPTICS LETTERS》, vol. 36, no. 15, 1 August 2011 (2011-08-01), XP001569569, DOI: doi:10.1364/OL.36.002865 * |
| ENRICO DIMITROW ET.AL: "Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis", 《EXPERIMENTAL DERMATOLOGY》, vol. 18, no. 6, 30 June 2009 (2009-06-30), XP055028070, DOI: doi:10.1111/j.1600-0625.2008.00815.x * |
| KARSTEN KOENIG ET.AL: "Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research", 《ADVANCED DRUG DELIVERY REVIEWS》, 13 April 2011 (2011-04-13) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297225A (en) * | 2014-09-29 | 2015-01-21 | 无限极（中国）有限公司 | Method for quickly detecting and imaging melanin in cells |
| 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 |
| WO2016180095A1 (en) * | 2015-05-08 | 2016-11-17 | 上海交通大学 | Method for detecting, in noninvasive in-vivo manner, skin damage induced by ultraviolet light, and detection device thereof |
| AU2016260153B2 (en) * | 2015-05-08 | 2019-04-04 | Jiangsu Kunhui Biotechnology Co., Ltd. | Method for detecting, in noninvasive in-vivo manner, skin damage induced by ultraviolet light, and detection device thereof |
| CN111134618A (en) * | 2015-05-08 | 2020-05-12 | 江苏坤辉生物科技有限公司 | Method and device for in vivo non-invasive detection of ultraviolet light induced skin injury |
| 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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| FR2982369A1 (en) | 2013-05-10 |
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| WO2013068943A1 (en) | 2013-05-16 |
| JP6147756B2 (en) | 2017-06-14 |
| FR2982369B1 (en) | 2016-05-06 |
| CN103930767B (en) | 2017-09-05 |
| KR20140090184A (en) | 2014-07-16 |
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