阅读说明：本技术 一种利用研究型正置显微成像系统定量分析斑马鱼黑色素的方法 (Method for quantitatively analyzing melanin of zebra fish by using research-type upright microscopic imaging system ) 是由 伦静雯 赵海山 苏奎 孙婷伟 吴少娟 郭清泉 于 2021-08-26 设计创作，主要内容包括：本发明属于生物测量技术领域,具体涉及一种利用研究型正置显微成像系统定量分析斑马鱼黑色素的方法；该方法主要是对斑马鱼体表进行成像,从而获得斑马鱼图像文件,然后对图像文件进行图像的相关性计算处理,即捕获清晰斑马鱼体表的黑色素分布图像,将其进行相关性计算处理来统计斑马鱼显微成像下体表黑色素的面积,即得斑马鱼黑色素的数据。本发明方法对实验对象本身不产生任何伤害,属于无接触测量,减少外界因素的影响,有效提高研究数据的可靠性。(The invention belongs to the technical field of biological measurement, and particularly relates to a method for quantitatively analyzing zebra fish melanin by using a research type upright microscopic imaging system; the method mainly comprises the steps of imaging the body surface of the zebra fish to obtain a zebra fish image file, then carrying out image correlation calculation processing on the image file, namely capturing a clear melanin distribution image of the zebra fish body surface, and carrying out correlation calculation processing on the image file to count the area of the melanin of the zebra fish body surface under microscopic imaging to obtain the data of the melanin of the zebra fish. The method does not cause any damage to the experimental object, belongs to non-contact measurement, reduces the influence of external factors, and effectively improves the reliability of research data.)

1. A method for quantitatively analyzing melanin of zebra fish by using a research type upright microscopic imaging system is characterized by sequentially comprising the following steps:

1) carrying out visualization processing on the measured zebra fish, and capturing zebra fish images by using a research-type upright microscope to obtain a group of zebra fish body surface open images;

2) then, carrying out correlation calculation processing on the group of images by utilizing Python and open CV, carrying out binarization processing on the original zebra fish image to convert the original zebra fish image into a gray image, and dividing the image into a plurality of rectangles; counting the rectangular area S in the zebra fish outline on each image_{Rectangle (Zebra fish)}And the rectangular area S occupied by melanin in the zebra fish outline_{Rectangle (melanin)}(ii) a Calculating the melanin area of each group of zebra fish according to the formula (1) to obtain a group of data, namely the melanin area data of the zebra fish,

wherein S_{Rectangle (Zebra fish)}Rectangular area, S, representing the contour of the zebra fish in the image_{Rectangle (melanin)}Representing the area of melanin in the zebrafish silhouette on the image.

2. The method for quantitatively analyzing the melanin of the zebra fish by using the research-type upright microscopic imaging system as claimed in claim 1, wherein the research-type upright microscopic imaging system is: axio imager.2 fluorescence upright microscope.

3. The method for quantitative analysis of melanin in zebra fish by using an upright microscope imaging system, as claimed in claim 1, wherein said upright microscope imaging system uses a 5 x magnifier to keep the parameters of the system consistent during shooting, so as to obtain a set of zebra fish open image files.

4. The method for quantitatively analyzing the melanin of the zebra fish by using the research-type upright microscopic imaging system as claimed in claim 1, wherein the correlation calculation processing method comprises the following steps: reading in the zebra fish image by using open CV, and carrying out graying and binarization on the image to convert the image into black and white chromaticities; then, acquiring image target frame information by using a Canny edge detector, wherein the detector detects an edge range in an image by using a multi-stage algorithm; then, converting melanin on the body surface of the zebra fish into black dots, cutting an image according to the target frame information, dividing the image into k multiplied by k rectangular frames, judging each rectangular frame, drying out abnormal dots according to the image background filtering rule, calculating the black and white area and abnormal values of the image, and filtering the abnormal values; numbering the filtered rectangular frames, and finally counting the area S of black points of each rectangular frame_{Rectangle (melanin)}。

5. The method for quantitatively analyzing the melanin of the zebra fish by using the research-type upright microscopic imaging system as claimed in claim 1, wherein the visualization process of the zebra fish to be measured specifically comprises the following steps:

1) culturing zebrafish embryos at 24-72 hpf in an incubator at a constant temperature of 28.5 ℃, treating the zebrafish embryos with drugs with different concentrations, and observing the condition of the melanin on the zebrafish body within a time period from 48hpf to the third week after fertilization;

2) controlling a constant experimental environment, connecting a research type upright microscope with computer equipment, and starting computer software;

3) placing carboxymethyl cellulose on a cover glass, placing the zebra fish to be detected on the carboxymethyl cellulose, fixing the zebra fish, then placing the cover glass with the zebra fish on an object stage of a microscope, and adjusting the required magnification to focus;

4) and acquiring a zebra fish body surface image by using computer software.

Technical Field

The invention belongs to the technical field of biological measurement, and particularly relates to a micro-imaging system and a zebra fish body surface melanin quantification method for biological in-vivo imaging.

Background

The zebra fish has the advantages of convenience in large-scale screening, convenience in feeding, short passage time, low price, capability of simply observing pigmentation process without complex experimental procedures due to the fact that black pigment is on the body surface of the zebra fish, and the like, and the change of the black pigment on the body surface of the zebra fish can be used for evaluating the effect of the whitening and freckle-removing raw material, so that the statistics of the black pigment on the body of the zebra fish is very important in researching and screening the raw material with the whitening component. At present, an absorbance spectroscopy method or a fluorescence spectroscopy method is generally adopted for quantifying melanin on the body surface of zebra fish, the steps of the methods are complicated, subjective factors have large influence, and interference on experiments is easily caused, so that melanin data is inaccurate.

1. Absorbance spectroscopy was used for melanin quantification: the method uses about 100 zebrafish embryos which are 9 to 48 hours after fertilization, and ultrasonic treatment is carried out in protein extracting solution. After centrifugation, the precipitate was dissolved in 1N NaOH at 100 ℃ for 10 minutes. The obtained supernatant was measured at an absorption spectrum having a wavelength of 490 nm. The method was published in 1996 by Busc a R, Bertolotto C, Ortonne JP, balloti R on JOURNAL OF biology CHEMISTRY (joural OF BIOLOGICAL chemisty): entitled "Inhibition of the Phosphositidylinositol 3-Kinase/p70S6-Kinase Pathway industries B16 Melanoma Cell Differentiation". Pub. No.: 31763 and 32482DEC 1996.

2. Fluorescence spectroscopy was used for quantification of melanin: the method uses ten embryos, ranging from 48hpf to 120hpf, with a steady increase in melanin production. Melanin fluoresces under oxidizing conditions with heat in an alkaline hydrogen peroxide solution, and the amount of melanin is collected at three selected time points. The method was manufactured in 2016 by Fernandes B, Matam T,d, Gomes A, Cavaco-Paulo A. published in pigment cell and melanoma research (PIGMENT CELL)&MELANOMA RESEARCH): the issue titled "Fluorescent qualification of melanin" is: 603-714NOV 2016.

However, the methods are complicated to operate and have many steps, and are destructive to zebra fish and have large subjective factor influence. In view of the points, the method for quantifying the melanin on the body surface of the zebra fish is simpler and more convenient, and brings more convenience to the research in the field of whitening cosmetics.

Disclosure of Invention

Aiming at the defects, the invention provides a non-contact, simple and convenient to operate and reliable-data quantitative analysis method for the zebra fish melanin.

Therefore, the technical scheme provided by the invention is as follows:

a method for quantitatively analyzing melanin of zebra fish by using a research type upright microscopic imaging system sequentially comprises the following steps:

1) carrying out visualization processing on the measured zebra fish, and capturing zebra fish images by using a research-type upright microscope to obtain a group of zebra fish body surface open images;

2) then, performing correlation calculation processing on the group of images by using a Python and open CV program, performing binarization processing on the original zebra fish image to convert the original zebra fish image into a gray image, and dividing the image into a plurality of rectangles; counting the rectangular area S in the zebra fish outline on each image_{Rectangle (Zebra fish)}And the rectangular area S occupied by melanin in the zebra fish outline_{Rectangle (melanin)}(ii) a Calculating the melanin area of each group of zebra fish according to the formula (1) to obtain a group of data, namely the melanin area data of the zebra fish,

wherein S_{Rectangle (Zebra fish)}Rectangular area, S, representing the contour of the zebra fish in the image_{Rectangle (melanin)}Representing the area of melanin in the zebrafish silhouette on the image.

Further, in the method for quantitatively analyzing melanin of zebra fish by using the research-type upright microscopic imaging system, the research-type upright microscopic imaging system comprises: axio imager.2 fluorescence upright microscope.

Further, in the method for quantitatively analyzing the melanin of the zebra fish by using the research-type upright microscopic imaging system, when the research-type upright microscopic imaging system is used for shooting, a magnifying glass with the magnification of 5 x is used, and the parameters of the system are kept consistent, so that a group of zebra fish open-bright image files are obtained.

Further, in the method for quantitatively analyzing the melanin of the zebra fish by using the research-type upright microscopic imaging system, the correlation calculation processing method comprises the following steps: reading in the zebra fish image by using open CV, and carrying out graying and binarization on the image to convert the image into black and white chromaticities; then, acquiring image target frame information by using a Canny edge detector, wherein the detector detects an edge range in an image by using a multi-stage algorithm; then, converting melanin on the body surface of the zebra fish into black dots, cutting an image according to the target frame information, dividing the image into k multiplied by k rectangular frames, judging each rectangular frame, drying out abnormal dots according to the image background filtering rule, calculating the black and white area and abnormal values of the image, and filtering the abnormal values; numbering the filtered rectangular frames, and finally counting the area S of black points of each rectangular frame_{Rectangle (melanin)}。

Further, the method for quantitatively analyzing the melanin of the zebra fish by using the research-type upright microscopic imaging system comprises the following steps of:

1) culturing zebrafish embryos at 24-72 hpf in an incubator at a constant temperature of 28.5 ℃, treating the zebrafish embryos with drugs with different concentrations, and observing the condition of the melanin on the zebrafish body within a time period from 48hpf to the third week after fertilization;

2) controlling a constant experimental environment, connecting a research type upright microscope with computer equipment, and starting computer software;

3) placing carboxymethyl cellulose on a cover glass, placing the zebra fish to be detected on the carboxymethyl cellulose, fixing the zebra fish, then placing the cover glass with the zebra fish on an object stage of a microscope, and adjusting the required magnification to focus;

4) and acquiring a zebra fish body surface image by using computer software.

The method utilizes a research-type upright microscope and combines a program related to image correlation calculation processing, has the characteristics of very simple, convenient, rapid and accurate quantification of the melanin on the body surface of the zebra fish, and highlights that the method provides non-contact measurement, does not bring any harm to organisms, and improves the accuracy and the reliability of experimental data. The method provides important application requirements and potential for the fields of living organisms, micro-illumination and imaging.

Drawings

FIG. 1 is a flow chart of the data processing method for image correlation calculation to quantify melanin on the body surface of zebra fish according to the invention.

Fig. 2 is a zebrafish image taken with an upright microscope.

Fig. 3 is a zebra fish silhouette image clipped using a rectangular frame.

Fig. 4 is an image a of the binarization processing. The B-picture is an image numbering zebra fish black specks.

FIG. 5 is a graph summarizing the effective black spots obtained by the filtration.

FIG. 6 is a statistical chart of the area ratio of zebrafish melanin under different administration concentrations of arbutin in panel A. And B is a blank group of zebra fish body surface images. The C group is the zebra fish body surface image with the concentration of 100mM arbutin group.

Detailed Description

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention in any way, and any limited number of modifications which can be made by anyone within the scope of the claims are still within the scope of the claims.

The most intuitive quantitative method for the melanin in the zebra fish body surface is to count the area of the melanin in the zebra fish body surface, so that the data of the melanin in the zebra fish can be essentially obtained by directly counting the areas of the melanin in the zebra fish body surfaces of different test groups.

When the melanin is quantified, the zebra fish to be detected needs to be subjected to body surface imaging firstly, and then a research type upright microscopic imaging system is used. During shooting, a magnifying glass with a certain multiple is used, the parameters of the system are kept consistent, so that a group of zebra fish spacious image files are obtained, and then image correlation calculation processing is carried out on the files:

and (4) reading the zebra fish image by using open CV, and carrying out graying and binarization on the image to convert the image into black and white chromaticity. And then acquiring the image target frame information by using a Canny edge detector, wherein the detector detects the edge range in the image by using a multi-stage algorithm. Then, the melanin on the zebra fish body surface is converted into black dots, the image is cut according to the target frame information to be divided into k multiplied by k rectangular frames, each rectangular frame is judged, abnormal dots are exposed through the image background filtering rule, and the black and white area of the image is calculated (namely S is obtained)_{Rectangle (Zebra fish)}) And filtering the abnormal value. Numbering the filtered rectangular frames, and finally counting the area S of black points of each rectangular frame_{Rectangle (melanin)}. We use S_{Rectangle (Zebra fish)}The sum of the areas of (A) and (B) gives the total area of the zebrafish, and S is used_{Rectangle (melanin)}The sum of (a) and (b) may yield the total area of melanin on the zebrafish, thus defined as:

in the above formula S_{Rectangle (Zebra fish)}Is the area of the rectangular frame of the zebra fish outline in the image, S_{Rectangle (melanin)}The area of the rectangular box of melanin within the zebra fish silhouette on the image.

In the operation process, the image meeting the requirements is selected as a target image, all the images to be processed are subjected to the above operation processing to obtain a group of data, and the melanin area of the zebra fish can be accurately and quickly reflected.

For example, a zebra fish picture (as shown in fig. 2) is selected, a rectangular frame (as shown in fig. 3) of the zebra fish outline in the picture is extracted, the area of the rectangular frame in the zebra fish outline is calculated, then black specks in the zebra fish outline are numbered (as shown in fig. 4 and 5), finally the area of the black specks under each number is calculated, the area of melanin can be obtained, the data of the melanin can be obtained according to the formula, and the group of data reflects the percentage of the melanin in the zebra fish, so that the melanin is visually quantified. And the method can clearly acquire the condition of each numbered black spot (as shown in figure 5). The measurement accuracy of the method depends on the stable imaging condition of a research-type upright microscope, and the research-type upright microscope can acquire clear zebra fish images and provide clear shooting conditions (constant color, natural light illumination, high-end chemical devices and configurable options) for melanin distribution.

Therefore, when the method provided by the invention is used, a clear shot image is required to be used, and the parameters of each captured image are ensured to be consistent, so that the reliability of the experimental result is improved. The specific procedures for quantifying the melanin of zebra fish can be summarized as follows:

1. zebrafish embryos are incubated in an incubator at a constant temperature of 28.5 ℃ for a period of time (typically 24hpf to 72hpf), treated with different conditions and observed for melanin in the zebrafish body, typically 48hpf to the third week after fertilization.

2. And controlling a constant experimental environment, connecting the research type upright microscope with equipment such as a computer, and starting computer software.

3. Placing carboxymethyl cellulose on a cover glass, placing the zebra fish to be detected on the carboxymethyl cellulose, fixing the zebra fish, then placing the cover glass with the zebra fish on a stage of a microscope, and adjusting the required magnification (for example, 5 times) for focusing.

4. And (3) acquiring a zebra fish body surface image by using computer software (as shown in figure 2).

5. Processing the collected images with pre-programmed software to obtain the melanin data map of zebra fish (as shown in figures 2, 3, 4 and 5)

The microscope model we used here is Axio Imager A2 from Zeiss

In order to better illustrate the advantages of the technical solution provided by the present application, the following provides an example of practical application of the technical solution provided by itself:

the effect of arbutin at different dosing concentrations on the melanin of zebrafish was measured:

1. wild type zebrafish embryos were cultured in a thermostated incubator at a constant temperature of 28.5 ℃.

2. And (4) building an experiment system required by shooting, and connecting a power supply detection device to determine whether the operation is normal.

3. Six groups of arbutin at different concentrations were used to treat 24hpf embryos to 72hpf, the first group was blank without drug treatment, and the following five groups were treated with drug at concentrations of 6.25mM, 12.5mM, 25mM, 50mM, 100mM, respectively.

4. Placing a zebra fish with a certain concentration on a cover glass with 4% of carboxymethyl cellulose, fixing the zebra fish on the cover glass, adjusting the posture of the zebra fish with the back facing upwards, enabling the whole zebra fish to be in a straight line state, and observing a clear melanin plaque on the body surface of the zebra fish by using a microscope with 5 x magnification.

5. Using ZEN software, exposure time was set: and 500s, collecting a top view of the distribution of the melanin of the zebra fish. As shown in fig. 2.

6. Naming the six groups of collected images, using the programmed program to process all image files in sequence, and calculating the area of melanin of each zebra fish, wherein the area is respectively

Ctrl group: 557751.5, 561046.5, 528218

6.25mM arbutin: 436457.5, 476063, 562656

12.5mM arbutin: 670607.5, 412138.5, 379182

25mM arbutin: 346250.5, 367243.5, 328673.5

50mM arbutin: 299224.5, 348216.5, 305681

100mM arbutin: 286122, 294449.5, 327264.5.

7. Counting the average value of melanin area under different administration concentration, Ctrl group: 549005.333333333, 6.25mM arbutin group: 491725.5, 12.5mM arbutin group: 487309.333333333, 25mM arbutin group: 347389.166666667, 50mM arbutin group: 317707.333333333, 100mM arbutin group: 302612, normalizing arbutin group with Ctrl group as 1, and drawing zebrafish melanin area ratio chart, as shown in fig. 6, the larger the value in graph A, the higher the melanin content, and the amount of melanin after arbutin treatment is obviously reduced and is concentration-dependent. In contrast, when the images are visually compared, the blank group is shown in the B image, and the administration group is shown in the C image, it is obvious that the melanin is obviously reduced after the arbutin treatment is performed.