CN114246548B - Device and system for detecting acne inflammation and application thereof - Google Patents
Device and system for detecting acne inflammation and application thereof Download PDFInfo
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Abstract
The invention discloses a device and a system for detecting acne inflammation and application thereof. The acne inflammation detection device provided by the invention comprises: the data receiving module is used for receiving images of different skin depths and fluorescence intensity information of the skin to be detected of the person to be detected; the data processing module is used for calculating the thickness of the epidermis and the fluorescence intensity difference value between the cell nucleus side and the cell edge side; the data storage module stores a judgment threshold value; a data comparison module; and a judging module. The invention establishes a noninvasive and living method for detecting the pathological changes of the acne inflammation through the TPEF imaging technology, provides a detection method for evaluating the acne inflammation, and provides a matched detection device and system to make up for the defect of researching the acne inflammation by the traditional method.
Description
Technical Field
The invention relates to a device and a system for detecting acne inflammation and application thereof.
Background
Acne is a chronic inflammatory disease of the pilosebaceous glands. Acne severely affects the quality of life of the patient. The cause and mechanism of acne is complex, but the inflammation is one of the most important factors of acne, and throughout the whole process of acne, the acne inflammation is not only the important cause of redness, swelling, itching and pain of the acne part, but also the important cause of pigmentation and acne scar formation, so how to detect skin acne is always a problem of dermatology and dermatology.
The existing evaluation method of acne on animals mainly focuses on visual observation and measurement of swelling degree of disease parts, the experimental error of the method is larger, the research is shallower, the deep research method mainly comprises the steps of observing pathological tissues, inflammatory factors and the like after taking materials, the methods all need detection after taking the pathological tissues, no wound and living bodies can be achieved, the method is complicated, the pathological process of the acne is difficult to track, and the state of living cells of each layer of epidermis can not be reflected.
Two-photon excited fluorescence (Two-Photon Excitation Fluorescence, TPEF) imaging techniques do not require any contrast agent to generate fluorescent signals to the epidermal endogenous fluorescent group nicotinamide adenine dinucleotide (phosphate) (nicotinamide adenine dinucleotide, NAD (P) H) and TPEF imaging techniques can track changes in these fluorescent signals in situ under pathological conditions.
Disclosure of Invention
In order to overcome the defects of the traditional method, the invention aims to establish an evaluation method for noninvasively detecting acne epidermitis and a device and a system for detecting acne inflammation, and provides a method for development of dermatology and dermatology.
In a first aspect, the invention claims an acne inflammation detection device.
The acne inflammation detection device claimed by the invention can comprise the following modules:
(1) A data receiving module; the data receiving module is configured to receive images of different skin depths and fluorescence intensity information at a skin to be measured of a subject.
(2) A data processing module; the data processing module is configured to receive the images of different skin depths and fluorescence intensity information transmitted by the data receiving module.
The data processing module comprises a data processing module 1 and a data processing module 2;
the data processing module 1 is configured to calculate an epidermis thickness value of the skin to be measured of the person to be measured from the images of the different skin depths and fluorescence intensity information.
Further, according to the TPEF imaging technique, the thickness from the occurrence of the granulosa cell NAD (P) H signal to the completion of basal lamina cell NAD (P) H signal collection starts from the uppermost layer of the epidermis as the entire epidermis thickness. In a specific embodiment of the invention, this is accomplished in particular by means of the self-contained software FV10-ASW V4.1 of a two-photon microscope.
The data processing module 2 is configured to extract a fluorescence distribution map of the epidermal particle layer cells from the images of the different depths and the fluorescence intensity information, and calculate a fluorescence intensity difference between the cell nucleus side and the cell edge side.
Further, the distance between the outer edge of the nucleus of the particle layer and the edge of the cell is halved, all halved points are connected to form a dividing line, and the difference between the average fluorescence intensity from the outer edge of the nucleus to the dividing line and the average fluorescence intensity from the edge of the cell to the dividing line is the difference between the fluorescence intensities at the side of the nucleus and the side of the edge of the cell. In a specific embodiment of the invention, this is accomplished in particular by means of the self-contained software FV10-ASW V4.1 of a two-photon microscope.
(3) A data storage module; the data storage module is configured to store a judgment threshold 1 and a judgment threshold 2.
The judging threshold 1 is a skin thickness judging threshold.
The judgment threshold 2 is a fluorescence intensity difference judgment threshold on the cell nucleus side and the cell edge side of the granular layer cell.
(4) A data comparison module; the data comparison module is configured to receive the epidermis thickness value of the skin to be tested of the person to be tested, which is sent by the data processing module 1, and call the judgment threshold value 1 from the storage module to compare with the epidermis thickness value of the skin to be tested of the person to be tested; and receiving the fluorescence intensity difference value of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be tested of the person to be tested, which is sent by the data processing module 2, and calling the judgment threshold 2 from the storage module to compare with the fluorescence intensity difference value of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be tested of the person to be tested.
(5) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition, judge whether the skin to be tested of the person to be tested meets the preset judging condition has or is candidate to have acne inflammation, judge whether the skin to be tested of the person to be tested does not meet the preset judging condition does not have or is candidate to have acne inflammation, and output the judging result.
Further, the predetermined determination condition may be: if the epidermis thickness value of the skin to be measured of the person to be measured is greater than the judgment threshold 1, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin to be measured of the person to be measured is greater than the judgment threshold 2, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
The judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judgment threshold 2 is a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin of a healthy person.
The data processing module may further comprise a data processing module 3 as required; the data processing module 3 is configured to extract fluorescence profiles of epidermal acantha and basal lamina cells from the images of different skin depths and fluorescence intensity information, and calculate a diameter value of the acantha cells and a diameter value of the basal lamina cells, respectively.
Further, the diameters of the fluorescence distribution of the acantha cells and the basal cells are measured by Image processing and analysis software Image J according to the extracted fluorescence distribution of the acantha cells and the basal cells.
Correspondingly, the data storage module is configured to store a judgment threshold 3 and a judgment threshold 4; the judgment threshold 3 is a diameter judgment threshold of the acantha layer cells, and the judgment threshold 4 is a diameter judgment threshold of the basal layer cells.
Correspondingly, the data comparison module is configured to receive the cell diameter value of the epidermal acantha layer at the skin to be tested of the person to be tested, which is sent by the data processing module 3, and call the judgment threshold 3 from the storage module to compare with the cell diameter value of the epidermal acantha layer at the skin to be tested of the person to be tested; and receiving the cell diameter value of the epidermal basal layer at the skin to be tested of the tested person, which is sent by the data processing module 4, and calling the judgment threshold value 4 from the storage module to compare with the cell diameter value of the epidermal basal layer at the skin to be tested of the tested person.
Accordingly, the predetermined determination condition may be: if the value of the epidermis thickness at the skin to be measured of the person to be measured is larger than the judging threshold 1, the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be measured of the person to be measured is larger than the judging threshold 2, the value of the cell diameter of the epidermis ratchet layer at the skin to be measured of the person to be measured is larger than the judging threshold 3, and the value of the cell diameter of the epidermis basal layer at the skin to be measured of the person to be measured is larger than the judging threshold 4, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
The judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judging threshold 2 is a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer of the skin of a healthy person; the judging threshold 3 is a cell diameter value of an epidermal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal layer of the skin of a healthy person; the judgment threshold 4 is a cell diameter value of an epidermal basal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal basal layer of the skin of a healthy person.
In the invention, the acne inflammation detection device can be realized by adopting a software program or hardware for realizing the functions of the corresponding modules, and the invention is not limited in any way.
In a preferred embodiment, the acne inflammation detection device of the present invention may further comprise a control module or controller for controlling the respective modules.
In the present invention, the functions of the control module or controller are the input of control data and the output of results, the transmission of data between the respective modules, and the data processing routine of the respective modules, and therefore any control device or controller such as a Central Processing Unit (CPU), a control chip for an industrial computer, or the like may be used as long as the above functions can be achieved.
It will be appreciated by those skilled in the art that the specific or preferred embodiments described above also apply to the various aspects herein below.
In a second aspect, the invention claims an acne inflammation detection system.
The acne inflammation detection system as claimed in the present invention may comprise a two-photon microscope for obtaining images of different skin depths and fluorescence intensity information at the skin to be tested of the tester and a control device, which may be configured or programmed to perform the steps of:
p1, receiving images of different skin depths and fluorescence intensity information of the skin to be detected of the person to be detected.
P2, calculating the epidermis thickness value of the skin to be measured of the person to be measured according to the images of the different skin depths and the fluorescence intensity information; extracting a fluorescence distribution map of the epidermal particle layer cells from the images with different depths and the fluorescence intensity information, and calculating a fluorescence intensity difference value of a cell nucleus side and a cell edge side;
p3, comparing the epidermis thickness value of the skin to be detected of the person to be detected with a judgment threshold 1, wherein the judgment threshold 1 is an epidermis thickness judgment threshold; comparing the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be detected of the person to be detected with a judgment threshold value 2, wherein the judgment threshold value 2 is the difference value judgment threshold value of the fluorescence intensity of the cell nucleus side and the cell edge side of the particle layer cell;
And P4, judging the comparison result according to a preset judgment condition, judging that the skin to be detected of the person to be detected which accords with the preset judgment condition has acne inflammation, judging that the skin to be detected of the person to be detected which does not accord with the preset judgment condition does not have acne inflammation, and outputting the judgment result.
Further, the predetermined determination condition may be: if the epidermis thickness value of the skin to be measured of the person to be measured is greater than the judgment threshold 1, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin to be measured of the person to be measured is greater than the judgment threshold 2, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
The judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judgment threshold 2 is a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin of a healthy person.
Further, in step P2, the method may further include: and extracting fluorescence distribution graphs of the epidermal acantha and basal lamina cells from the images of different skin depths and the fluorescence intensity information, and respectively calculating the diameter value of the acantha cells and the diameter value of the basal lamina cells.
Accordingly, in step P3, the method may further include: comparing the cell diameter value of the epidermal acantha layer at the skin to be detected of the person to be detected with a judgment threshold value 3, wherein the judgment threshold value 3 is the diameter judgment threshold value of the acantha layer cell; and comparing the cell diameter value of the basal layer of the epidermis at the skin to be detected of the person to be detected with a judgment threshold value 4, wherein the judgment threshold value 4 is the diameter judgment threshold value of basal layer cells.
Accordingly, the predetermined determination condition may be: if the value of the epidermis thickness at the skin to be measured of the person to be measured is larger than the judging threshold 1, the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be measured of the person to be measured is larger than the judging threshold 2, the value of the cell diameter of the epidermis ratchet layer at the skin to be measured of the person to be measured is larger than the judging threshold 3, and the value of the cell diameter of the epidermis basal layer at the skin to be measured of the person to be measured is larger than the judging threshold 4, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
The judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judging threshold 2 is a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer of the skin of a healthy person; the judging threshold 3 is a cell diameter value of an epidermal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal layer of the skin of a healthy person; the judgment threshold 4 is a cell diameter value of an epidermal basal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal basal layer of the skin of a healthy person.
In the above system, the control means may be any computer capable of executing the above steps, such as a Personal Computer (PC), an industrial control computer, or the like.
Further, the system may also include an input device and an output device; the input device is configured to input images of the different skin depths and fluorescence intensity information from the two-photon microscope; the output device is configured to output the determination result.
In the present invention, the input device may be any type of input device commonly used for various types of data, such as a keyboard, a mouse, a camera, a scanner, a light pen, a handwriting input board, a joystick, a voice input device, and the like. The output device may be any type of output device commonly used for various types of data, such as a display, a printer, a plotter, an image output system, a voice output system, a magnetic recording device, and the like.
In a third aspect, the invention claims a method of using the system described above.
The method for using the system as claimed in the invention can comprise the following steps: detecting images of different skin depths and fluorescence intensity information of the skin to be detected of a person to be detected by using a two-photon microscope; the control device in the system is used for executing the steps, and determining whether the skin to be tested of the testee has acne inflammation or not according to the judging result.
In a fourth aspect, the present invention claims a computer readable storage medium storing a computer program for performing the steps of:
p1, receiving images of different skin depths and fluorescence intensity information of the skin to be detected of the person to be detected.
P2, calculating the epidermis thickness value of the skin to be measured of the person to be measured according to the images of the different skin depths and the fluorescence intensity information; extracting a fluorescence distribution map of the epidermal particle layer cells from the images with different depths and the fluorescence intensity information, and calculating a fluorescence intensity difference value of a cell nucleus side and a cell edge side;
p3, comparing the epidermis thickness value of the skin to be detected of the person to be detected with a judgment threshold 1, wherein the judgment threshold 1 is an epidermis thickness judgment threshold; comparing the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be detected of the person to be detected with a judgment threshold value 2, wherein the judgment threshold value 2 is the difference value judgment threshold value of the fluorescence intensity of the cell nucleus side and the cell edge side of the particle layer cell;
and P4, judging the comparison result according to a preset judgment condition, judging whether the skin to be tested of the tested person meets the preset judgment condition has or is candidate to have acne inflammation, judging whether the skin to be tested of the tested person does not meet the preset judgment condition does not have or is candidate to have acne inflammation, and outputting the judgment result.
Further, the predetermined determination condition may be: if the epidermis thickness value of the skin to be detected of the person to be detected is greater than the judging threshold 1, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin to be detected of the person to be detected is greater than the judging threshold 2, acne inflammation exists at the skin to be detected of the person to be detected; otherwise, the skin to be tested of the testee does not have acne inflammation.
The judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judgment threshold 2 is a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin of a healthy person.
Further, in step P2, the method may further include: and extracting fluorescence distribution graphs of the epidermal acantha and basal lamina cells from the images of different skin depths and the fluorescence intensity information, and respectively calculating the diameter value of the acantha cells and the diameter value of the basal lamina cells.
Accordingly, in step P3, the method may further include: comparing the cell diameter value of the epidermal acantha layer at the skin to be detected of the person to be detected with a judgment threshold value 3, wherein the judgment threshold value 3 is the diameter judgment threshold value of the acantha layer cell; and comparing the cell diameter value of the basal layer of the epidermis at the skin to be detected of the person to be detected with a judgment threshold value 4, wherein the judgment threshold value 4 is the diameter judgment threshold value of basal layer cells.
Accordingly, the predetermined determination condition may be: if the value of the epidermis thickness at the skin to be measured of the person to be measured is larger than the judging threshold 1, the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be measured of the person to be measured is larger than the judging threshold 2, the value of the cell diameter of the epidermis ratchet layer at the skin to be measured of the person to be measured is larger than the judging threshold 3, and the value of the cell diameter of the epidermis basal layer at the skin to be measured of the person to be measured is larger than the judging threshold 4, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
The judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judging threshold 2 is a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer of the skin of a healthy person; the judging threshold 3 is a cell diameter value of an epidermal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal layer of the skin of a healthy person; the judgment threshold 4 is a cell diameter value of an epidermal basal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal basal layer of the skin of a healthy person.
In a fifth aspect, the invention claims the use of a two-photon microscope for detecting acne inflammation or for the preparation of a product for detecting acne inflammation.
In a sixth aspect, the present invention claims a screening method for an acne inflammation preventing and treating drug.
The screening method of the acne inflammation preventing and treating medicine claimed by the invention can comprise the following steps:
(a1) Experimental group: administering a test drug to a test animal while or prior to inoculating the skin of the test animal with propionibacterium acnes (Propionibacterium acnes, p.acnes);
control group: inoculating propionibacterium acnes to the skin of the test animal;
(a2) Detecting the cell diameter of the epidermal layer and/or basal layer of the skin of the tested animal where the tested animal is inoculated with propionibacterium acnes on the next day after the skin of the tested animal is inoculated with propionibacterium acnes in the test group and the control group, and if the cell diameter of the epidermal layer and/or basal layer of the skin of the tested animal where the tested animal is inoculated with propionibacterium acnes in the test group is significantly smaller than the cell diameter of the epidermal layer and/or basal layer of the skin of the tested animal where the tested animal is inoculated with propionibacterium acnes in the control group, the drug to be tested is or is candidate to be an acne inflammation preventing drug; otherwise, the medicine to be detected is not an acne inflammation prevention and treatment medicine.
In step (a 2), the cell diameters of the epidermal acantha and/or basal lamina of the skin where the test animals are inoculated with propionibacterium acnes in the test and control groups can be achieved by a two-photon excitation fluorescence microscopy imaging technique.
Further, in step (a 2), it may further include: detecting the skin thickness of the skin of the test animal where the test animal is inoculated with propionibacterium acnes (which can be achieved by two-photon excitation fluorescence microscopy imaging technique) in the experimental group and the control group within the second to eight days after inoculating the skin of the test animal with propionibacterium acnes; and/or detecting the difference in fluorescence intensity of the nucleus side and the cell edge side of the epidermis particle layer of the skin where the test animal is inoculated with propionibacterium acnes in the experimental group and the control group by a two-photon excitation fluorescence microscopy imaging technique within the second to fifteen days after the skin of the test animal is inoculated with propionibacterium acnes. The test agent is or is candidate as an acne inflammation preventing agent if the skin thickness of the skin of the test animal inoculated with propionibacterium acnes in the experimental group is significantly smaller than the skin thickness of the skin of the test animal inoculated with propionibacterium acnes in the control group, and/or if the difference in fluorescence intensity between the cell nucleus side and the cell edge side of the skin of the test animal inoculated with propionibacterium acnes in the experimental group is significantly smaller than the difference in fluorescence intensity between the cell nucleus side and the cell edge side of the skin of the test animal inoculated with propionibacterium acnes in the control group; otherwise, the medicine to be detected is not an acne inflammation prevention and treatment medicine.
Wherein the test animal may be a mouse. The propionibacterium acnes can be inoculated to a site such as the ear of a mouse.
In a specific embodiment of the invention, the propionibacterium acnes is specifically p.acnes standard strain ATCC6919.
In the above aspects, when the two-photon excitation fluorescence microscopy imaging technique is used to detect the epidermis thickness of the skin to be detected, the fluorescence intensities of the nucleus side and the cell edge side of the epidermis particle layer, and the cell diameters of the epidermis ratchet layer and/or the basal layer, the following parameters can be used: the epidermal tissue is scanned by a 25-time water immersion microscope objective with the numerical aperture of 1.05, the steps are 5 mu m, the image acquisition pixels are 1 024 pixels multiplied by 1 024 pixels, the acquisition speed is 4 mu m pixels -1, and the light is received by a filter plate with the wavelength of 420-460 nm.
In the above aspects, the subject may be a suspected patient of acne identified by visual inspection and/or measurement of the extent of swelling at the site of the disorder.
The invention detects the change of the propionibacterium acnes caused by the epidermitis by a Two-photon excitation fluorescence (Two-Photon Excitation Fluorescence, TPEF) imaging technology. P.acnes in the logarithmic growth phase was injected into the ears of mice, and the thickness of the epidermis, the distribution of nicotinamide adenine dinucleotide (phosphoric acid) (nicotinamide adenine dinucleotide, NAD (P) H) in the granulosa layer cells, and the change in the diameters of the cells of the acantha layer and basal layer were detected by TPEF imaging technique, while the thickness of the ears and epidermis were detected by vernier calipers and HE staining method. TPEF imaging technology finds that before molding, the thickness of epidermis is thinner, the signals of granular layer cells NAD (P) H are uniformly distributed in cytoplasm, the diameters of the cells of the acantha layer and the basal layer are smaller, after P.acnes are injected, the thickness of epidermis is obviously increased, the granular layer cells NAD (P) H are concentrated at the nucleus, and the diameters of the cells of the acantha layer and the basal layer are obviously increased. Vernier calipers and HE staining found increased ear and epidermis thickness after p.acnes stimulation. The invention hopes to establish a noninvasive and living method for detecting acne inflammation, and provides a matched detection device and system, which provides reference basis for evaluating acne inflammation and skin acne cosmetology development.
Drawings
Fig. 1 shows the variation in the appearance and thickness of the ear. (a) is a change in appearance of the ear at different times; (b) is the variation of ear thickness at different times.
FIG. 2 is a graph showing the effects of TPEP imaging techniques and HE staining to detect P.acnes on skin thickness. (a) detecting the thickness of the epidermis for a TPEP imaging technique; (b) detecting the thickness of the epidermis for HE staining; (c) detecting an epidermis thickness analysis for a TPEP imaging technique; (d) detecting skin thickness analysis for HE staining.
FIG. 3 is a graph showing the effect of TPEF imaging technique on the distribution of fluorescent signals of NAD (P) H in granulosa cells. (a) Imaging the granulosa layer cells NAD (P) H for TPEF imaging techniques; (b) fluorescent signal analysis of the granulosa layer cells NAD (P) H.
Fig. 4 is a graph of the effects of TPEF imaging technology on the cell size of the stratum spinosum and stratum basale. (a) imaging the cells of the stratum spinosum by TPEF imaging technique; (b) imaging the basal layer cells using TPEF imaging techniques; (c) is an analysis of acantha cell diameter; (d) basal layer cell diameter analysis.
Fig. 5 is a computer flow chart for implementing the acne inflammation detection of the present invention.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 acne inflammation detection
1. Material and apparatus
Acne inflammation detection system: including acne inflammation detection devices (see above) and two-photon scanning microscopes (Olympus corporation).
Paraffin microtomes, olympus corporation; a small animal anesthesia machine, shenzhen Ruiwod life technologies Co., ltd; inverted microscope, olympus; an oxygen generating pouch, an anaerobic indicator, mitsubishi gas chemical company, japan; BHI medium, sigma, usa; agar american Sigma; depilatory cream, lijieshua (China) Limited.
ICR mice; beijing Vitolihua laboratory animal technology Co., ltd; propionibacterium acnes (Propionibacterium acnes, p.acnes) standard strain (ATCC 6919), available from the microbiological institute of Cantonese province.
2. Experimental method
1. P.acnes culture
Anaerobic culturing P.acnes in BHI solid culture medium, collecting P.acnes in logarithmic phase after 3 days, regulating bacterial concentration to 3×10 8CFU·L-1, cleaning with PBS for 3 times, and preparing for use.
2. Animal model building
15 ICR mice, male, weighing 25-30g, were adapted for 1 week post-molding. The present experiment adopts the commonly used modeling method [Nakatsuji T,Kao M C,Fang J Y,et al.Antimicrobial property of lauric acid against Propionibacterium acnes:its therapeutic potential for inflammatory acne vulgaris[J].The Journal of investigative dermatology,2009,129(10):2480-2488.],P.acnes of injecting the P.acnes into the ears to cause acne, which is an important factor for causing the acne. Animals were anesthetized on the day of molding, and after deep anesthesia, ears were injected at a concentration of 3×10 8CFU·L-1 p.acnes 30 μl, and ears were observed after 1 day if redness and swelling clearly indicated that the model construction was successful.
In the experiment, the injection of the equal volume PBS was used as a negative control group, and the pre-experiment in the early stage of the experiment found that the injection of the equal volume PBS had no change in the ear, so that the following experiment was directly performed by using the pre-injection (Day 0) as a negative control.
3. Vernier caliper for detecting ear thickness variation
Before molding (Day 0, i.e., molding was not performed on the same Day), 1 Day after molding (Day 1), 7 days (Day 7), 14 days (Day 14), the change in ear thickness was detected by a vernier caliper, and the ears were photographed.
4. TPEP imaging technique
And respectively fixing the mice on a two-photon microscope stage after being anesthetized by isoflurane at Day 0,1,7 and 14, scanning epidermal tissues by adopting a 25-time water immersion microscope objective with a numerical aperture of 1.05, stepping by 5 mu m, collecting the pixels 1 024pixel x 1 024pixel at a collecting speed of 4 mu m pixel -1, and respectively analyzing the thickness of the epidermis, the fluorescence change of cells of a particle layer, the diameters of cells of a spine layer and a basal layer after collecting the cells by a filter of 420-460 nm.
5. HE staining
3 Mice were anesthetized at Day 0,1,7 and 14, ear pathological tissue was obtained after deep anesthesia, the tissue was fixed with 4% paraformaldehyde for 48 hours, and after trimming, dehydrating, transparentizing, embedding the tissue, cutting into 6 μm thick sections, further dewaxing, staining, dehydrating, transparentizing and sealing, and finally observing the epidermis thickness by an inverted microscope.
6. Data processing
All data were calculated by GRAPHPAD PRISM software; all data were statistically processed using SPSS17.0 software, and the group comparisons were statistically significant using one-way anova, expressed as mean+ -SD, with P <0.05 as the difference.
2. Results and discussion
1. Acne inflammation model and ear thickness variation
To observe the changes in the ears before and after p.acnes injection, mice ears were photographed at Day 0,1,7 and 14, respectively, and ear thickness was measured, and these time points represent the entire course of acne from no onset to severe onset, followed by slow remission, respectively. The results found that the mice had thinner ears and whiter color before p.acnes injection. When day1 was injected, the ear red swelling was evident in the mice (fig. 1 (a)), and the thickness was significantly increased (P < 0.001). Over time, at Day7 and Day 14, the ear redness and swelling gradually decreased (fig. 1 (a)), but the ear thickness was still significantly thickened (P <0.001 ) compared to Day 0.
2. Variation of skin thickness
The nonlinear spectrum resolution imaging technology generated by TPEF is an emerging imaging technology, and can simultaneously obtain images and spectrum information of different depths of biological tissues by combining two-photon excitation fiber technology and spectrum measurement technology. The two-photon imaging has the characteristics of high sensitivity, high resolution and low killers, and can be used for noninvasively and living body tracking the dynamic process of endogenous fluorescent groups in living cell tissues. NAD (P) H, an endogenous fluorescent signal, is mainly produced by biological cells during metabolism, and its basic physiological function is to maintain cell growth, differentiation and energy metabolism [21], and in epidermal tissue, it is distributed in living cells such as granular layer, acanthal layer and basal layer, so that it can reflect the change of epidermal thickness by detecting NAD (P) H.
The thickness of the ear epidermis is detected through TPEP imaging technology and HE staining, and according to the TPEF imaging technology, the thickness from the signal of granular layer cells NAD (P) H to the signal of basal layer cells NAD (P) H is acquired and is taken as the whole epidermis thickness (which is finished through self-contained software FV10-ASW V4.1 of a two-photon microscope). TPEP imaging technical results show that Day 0 has a thinner normal epidermis thickness, which is 20-25 μm. At Day 1, the skin thickness increased significantly (fig. 2 (a) and (c)), with statistical significance (P < 0.01) compared to Day 0. At Day 7, the skin thickness was thinned (fig. 2 (a) and (c)), but the skin thickening was still significant (P < 0.01) compared to Day 0. At Day 14, the skin thickness gradually decreased (fig. 2 (a) and (c)), with no statistical significance (P > 0.05) compared to Day 0. HE staining results showed that Day 1 and Day 7 had significantly higher skin thickness than Day 0 (fig. 2 (b) and (d)) (P < 0.01), and Day 14 had significantly reduced skin thickness but still higher skin thickness than Day 0 (fig. 2 (b) and (d)) (P < 0.05). Both TPEP imaging technique and HE staining results showed that the skin thickness increased most significantly after p.acnes injection for 1 day, with the skin thickness gradually decreasing over time.
The invention discovers that the normal skin thickness of the mice is basically consistent with the thickness of Jonathan A Palero[Palero J A,De B H S,Van D P D H A,et al.In vivo nonlinear spectral imaging in mouse skin[J].Optics express,2006,14(10):4395-4402.] detected epidermis by TPEF imaging technology, but is the same as that of the literature [Jiang Xingshan,ZhuoShuangmu,Xu Ren'an,et al.Multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation[J].Scanning,2012,34(3):170-173.Jonathan A,S,De B.et al.Gerritsen Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues[J].Biophysical Journal,2007,93(3):992-1007.] The results of the detection in (a) are inconsistent, and the reason for this is found that the present invention detects the thickness of living cells including the particle layer, the spine layer and the basal layer, and document [Jiang Xingshan,ZhuoShuangmu,Xu Ren'an,et al.Multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation[J].Scanning,2012,34(3):170-173.Jonathan A,/>S,De B.et al.Gerritsen Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues[J].Biophysical Journal,2007,93(3):992-1007.] In the test, the stratum corneum and the upper part of the hair follicle of the epidermis are included in addition to the living cells, so that the total thickness is not completely uniform. In short, not only the thickness of the epidermis closely related to the severity of skin inflammation can be detected by the TPEF imaging technique, but also the states of the living cells of each layer of the epidermis can be clearly presented, which overcomes the defects that HE staining cannot detect the living cells and the cells of each layer cannot be detected.
3. Variation of epidermal particle layer cell fluorescence signal distribution
In order to study pathological changes of cells of the epidermis particle layer, the fluorescent distribution of NAD (P) H of the cells of the epidermis particle layer is detected by a TPEF imaging technology, and meanwhile, the fluorescent intensity changes of a cell nucleus side area and a cell edge side area are detected and analyzed as detection indexes. The distance between the outer edge of the nucleus of the particle layer and the edge of the cell is halved, all halved points are connected to form a parting line, and the difference between the average fluorescence intensity from the outer edge of the nucleus to the parting line and the average fluorescence intensity from the edge of the cell to the parting line is the difference between the fluorescence intensities of the side of the nucleus and the side of the cell edge (which is finished by self-contained software FV10-ASW V4.1 of a two-photon microscope). NAD (P) H of the normal mouse epidermis particle layer is uniformly distributed in cytoplasm, and the fluorescence change value (i.e., the difference between fluorescence intensities at the nucleus side and the cell edge side) is small. In Day1, the TPEF signal is concentrated around the nucleus (fig. 3 (a)), and the perinuclear fluorescent signal is significantly increased (fig. 3 (b), P < 0.01) compared to Day 0. At Day7, the TPEF signal was gradually developed in the region farther from the nucleus (fig. 3 (a)), but the fluorescence signal change (i.e., the difference in fluorescence intensity between the nucleus side and the cell edge side) was significantly increased (fig. 3 (b), P < 0.05) compared to Day 0. In Day14, although the fluorescence intensity of the cell edge side region gradually increased (fig. 3 (a)), the fluorescence signal change value (i.e., the difference in fluorescence intensity between the cell nucleus side and the cell edge side) still significantly increased (fig. 3 (b), P < 0.05) compared to Day 0. In summary, we conclude that the distribution of epidermal granulosa cell NAD (P) H is significantly altered in P.acnes-induced inflammatory changes.
4. Changes in epidermal acantha and basal lamina cells
To study the change in the size of the epidermal acantha cells, the change in acantha cell diameter was observed by detecting NAD (P) H fluorescent signals in the acantha by TPEF imaging technique. And measuring the diameters of the fluorescence distribution of the cells of the stratum spinosum and the stratum basale at different times before and after the molding by using Image processing and analysis software Image J according to the extracted fluorescence distribution map of the cells of the stratum spinosum and the stratum basale). Normal skin acantha cell diameter is small (fig. 4 (a) and (c)). Day1 spinous layer cell diameter increased significantly after p.acnes injection by 1.23 fold over Day 0 spinous layer cell diameter (fig. 4 (a) and (c), P < 0.01). At Day7, the acantha cell diameter was larger than Day 0 normal acantha cells, but not statistically significant (P > 0.05 in fig. 4 (a) and (c)). At Day14, the acantha cell diameter gradually decreased, substantially in accordance with Day 0 acantha cell diameter (fig. 4 (a) and (c), P > 0.05).
Changes in basal layer cell diameter were detected and analyzed by TPEF imaging techniques. Day 0 basal lamina has a smaller cell diameter (fig. 4 (b) and (d)). Day1 basal lamina cells were 1.28 fold more than normal basal lamina cells of skin, statistically significant (fig. 4 (b) and (d), P < 0.05). Although Day7 basal lamina cells had larger diameters than Day 0 normal epidermal basal lamina cells over time, there was no statistical significance (P > 0.05 in fig. 4 (b) and (d)). At Day14, there was no difference in basal cell diameter from Day 0 normal basal cell diameter (P > 0.05 in fig. 4 (b) and (d)). These results indicate that the diameter of the cells of the stratum spinosum and basal lamina can be significantly increased after 1 day of p.acnes stimulation, which lays a foundation for the later screening of acne inflammation preventing and treating drugs by evaluating the changes of the drugs on the diameters of the cells of the stratum spinosum and basal lamina.
According to the invention, pathological changes of epidermic inflammation caused by P.acnes are detected in a noninvasive and living way through a TPEF imaging technology, and as a result, normal epidermis is thinner, granular layer cells NAD (P) H are uniformly distributed in cytoplasm, the cells of the stratum spinosum and stratum basale are smaller, the epidermis is thickened after P.acnes are stimulated, the granular layer cells NAD (P) H are intensively distributed around the cell nucleus, and the cells of the stratum spinosum and stratum basale become larger, so that the distribution of the granular layer cells NAD (P) H, the sizes of the cells of the stratum spinosum and the stratum basale can be used as important indexes for evaluating acne inflammation in future. The invention detects through the traditional vernier caliper and HE dyeing method at the same time, and discovers that the thickness of the ear and the epidermis is thickened after P.acnes stimulation, but the pathological state of each layer of cells cannot be characterized. In a word, the invention hopes to establish a noninvasive and living method for detecting the pathological changes of the acne inflammation through TPEF imaging technology, provides a detection method for evaluating the acne inflammation, and provides a matched detection device and system to make up for the defect of researching the acne inflammation by the traditional method.
Fig. 5 is a computer flow chart for implementing the acne inflammation detection of the present invention.
In step S1, receiving images of different skin depths and fluorescence intensity information of the skin to be tested of a person to be tested;
In step S2, either step S21 or step S22 is performed;
In step S21, calculating the epidermis thickness value at the skin to be measured of the subject, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermal particle layer cells;
in step S22, calculating the thickness value of the epidermis at the skin to be measured of the subject, the difference in fluorescence intensity between the cell nucleus side and the cell edge side of the cells of the epidermis particle layer, and the diameter value of the cells of the epidermis ratchet layer and the basal layer;
In step S3, the index value calculated in step S2 is compared with a corresponding threshold value, if both the index values are greater than the threshold value, it is determined that the acne inflammation exists or is candidate to exist, otherwise, it is determined that the acne inflammation does not exist or is candidate to exist.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (7)
1. An acne inflammation detection device comprising the following modules:
(1) A data receiving module; the data receiving module is configured to receive images of different skin depths and fluorescence intensity information of the skin to be tested of the tested person;
(2) A data processing module; the data processing module is configured to receive the images of different skin depths and fluorescence intensity information transmitted by the data receiving module;
the data processing module comprises a data processing module 1 and a data processing module 2;
The data processing module 1 is configured to calculate an epidermis thickness value at the skin to be measured of the person to be measured according to the images of the different skin depths and fluorescence intensity information; the data processing module 2 is configured to extract a fluorescence distribution map of the epidermal particle layer cells from the images of the different skin depths and the fluorescence intensity information, and calculate a fluorescence intensity difference value between the cell nucleus side and the cell edge side;
(3) A data storage module; the data storage module is configured to store a judgment threshold value 1 and a judgment threshold value 2;
The judging threshold 1 is a skin thickness judging threshold;
the judging threshold 2 is a fluorescence intensity difference judging threshold of the nucleus side and the cell edge side of the granular layer cells;
(4) A data comparison module; the data comparison module is configured to receive the epidermis thickness value of the skin to be tested of the person to be tested, which is sent by the data processing module 1, and call the judgment threshold value 1 from the storage module to compare with the epidermis thickness value of the skin to be tested of the person to be tested; and receiving the difference value of fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be tested of the person to be tested, which is sent by the data processing module 2, and calling the judgment threshold 2 from the storage module to compare with the difference value of fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be tested of the person to be tested;
(5) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition, judge whether the skin to be tested of the person to be tested meets the preset judging condition has or is candidate to have acne inflammation, judge whether the skin to be tested of the person to be tested does not meet the preset judging condition does not have or is candidate to have acne inflammation, and output the judging result;
The predetermined determination condition is: if the epidermis thickness value of the skin to be measured of the person to be measured is greater than the judgment threshold 1, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin to be measured of the person to be measured is greater than the judgment threshold 2, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
2. The apparatus according to claim 1, wherein: the judging threshold value 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judgment threshold 2 is a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin of a healthy person.
3. The apparatus according to claim 1, wherein: the data processing module further comprises a data processing module 3; the data processing module 3 is configured to extract fluorescence distribution diagrams of epidermal acantha and basal lamina cells from the images of different skin depths and fluorescence intensity information, and calculate diameter values of the acantha cells and the basal lamina cells respectively;
the data storage module is configured to store a judgment threshold 3 and a judgment threshold 4; the judging threshold 3 is a diameter judging threshold of the stratum spinosum cells, and the judging threshold 4 is a diameter judging threshold of the stratum basale cells;
The data comparison module is configured to receive the cell diameter value of the epidermal layer at the skin to be tested of the person to be tested, which is sent by the data processing module 3, and call the judgment threshold value 3 from the storage module to compare with the cell diameter value of the epidermal layer at the skin to be tested of the person to be tested; and receiving the cell diameter value of the epidermal basal layer at the skin to be tested of the tested person, which is sent by the data processing module 4, and calling the judgment threshold value 4 from the storage module to compare with the cell diameter value of the epidermal basal layer at the skin to be tested of the tested person.
4. A device according to claim 3, characterized in that: the predetermined determination condition is: if the value of the epidermis thickness at the skin to be measured of the person to be measured is larger than the judging threshold 1, the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be measured of the person to be measured is larger than the judging threshold 2, the value of the cell diameter of the epidermis ratchet layer at the skin to be measured of the person to be measured is larger than the judging threshold 3, and the value of the cell diameter of the epidermis basal layer at the skin to be measured of the person to be measured is larger than the judging threshold 4, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
5. The apparatus according to claim 3 or 4, wherein: the judging threshold 1 is a skin thickness value of the skin to be detected of the person to be detected in a healthy state or a skin thickness value of the skin of a healthy person; the judging threshold 2 is a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer when the skin to be tested of the person to be tested is in a healthy state, or a difference value of fluorescence intensity of a cell nucleus side and a cell edge side of the epidermis particle layer of the skin of a healthy person; the judging threshold 3 is a cell diameter value of an epidermal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal layer of the skin of a healthy person; the judgment threshold 4 is a cell diameter value of an epidermal basal layer of the skin to be tested of the person to be tested in a healthy state or a cell diameter value of an epidermal basal layer of the skin of a healthy person.
6. An acne inflammation detection system comprising a two-photon microscope and a control device, the control device being configurable or programmable to perform the steps of:
p1, receiving images of different skin depths and fluorescence intensity information of the skin to be detected of a person to be detected;
p2, calculating the epidermis thickness value of the skin to be measured of the person to be measured according to the images of the different skin depths and the fluorescence intensity information; extracting a fluorescence distribution map of the epidermal particle layer cells from the images of different skin depths and the fluorescence intensity information, and calculating a fluorescence intensity difference value of a cell nucleus side and a cell edge side;
p3, comparing the epidermis thickness value of the skin to be detected of the person to be detected with a judgment threshold 1, wherein the judgment threshold 1 is an epidermis thickness judgment threshold; comparing the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be detected of the person to be detected with a judgment threshold value 2, wherein the judgment threshold value 2 is the difference value judgment threshold value of the fluorescence intensity of the cell nucleus side and the cell edge side of the particle layer cell;
P4, judging the comparison result according to a preset judgment condition, judging whether the skin to be tested of the tested person meets the preset judgment condition has or is candidate to have acne inflammation, judging whether the skin to be tested of the tested person does not meet the preset judgment condition does not have or is candidate to have acne inflammation, and outputting a judgment result;
The predetermined determination condition is: if the epidermis thickness value of the skin to be measured of the person to be measured is greater than the judgment threshold 1, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin to be measured of the person to be measured is greater than the judgment threshold 2, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
7. A computer readable storage medium storing a computer program for performing the steps of:
p1, receiving images of different skin depths and fluorescence intensity information of the skin to be detected of a person to be detected;
p2, calculating the epidermis thickness value of the skin to be measured of the person to be measured according to the images of the different skin depths and the fluorescence intensity information; extracting a fluorescence distribution map of the epidermal particle layer cells from the images of different skin depths and the fluorescence intensity information, and calculating a fluorescence intensity difference value of a cell nucleus side and a cell edge side;
p3, comparing the epidermis thickness value of the skin to be detected of the person to be detected with a judgment threshold 1, wherein the judgment threshold 1 is an epidermis thickness judgment threshold; comparing the difference value of the fluorescence intensity of the cell nucleus side and the cell edge side of the epidermis particle layer at the skin to be detected of the person to be detected with a judgment threshold value 2, wherein the judgment threshold value 2 is the difference value judgment threshold value of the fluorescence intensity of the cell nucleus side and the cell edge side of the particle layer cell;
P4, judging the comparison result according to a preset judgment condition, judging whether the skin to be tested of the tested person meets the preset judgment condition has or is candidate to have acne inflammation, judging whether the skin to be tested of the tested person does not meet the preset judgment condition does not have or is candidate to have acne inflammation, and outputting a judgment result;
The predetermined determination condition is: if the epidermis thickness value of the skin to be measured of the person to be measured is greater than the judgment threshold 1, and the fluorescence intensity difference between the cell nucleus side and the cell edge side of the epidermis particle layer of the skin to be measured of the person to be measured is greater than the judgment threshold 2, the skin to be measured of the person to be measured has or is candidate to have acne inflammation; otherwise, the skin of the tested person has no or candidate acne inflammation.
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