Method for automatic processing of taxis experiment and image data of model organism nematodes

Abstract

The invention discloses a method for automatically processing image data of a nematode tropism experiment and belongs to the technical field of biological experiments and image data processing. The invention solves the technical problem of how to realize automatic processing of image data of a nematode tropism experiment. The technical solution is as follows: different areas are divided on an NGM culture medium of the model organism nematode and volatile substances are dripped in the designated areas; 50-100 young adult nematodes treated with chemicals are spread in the center of an assay plate; after being placed indoors in the dark for 0.5-1.5 hours, experimental images are photographed under a stereomicroscope; experimental areas that actually need to be analyzed are identified from the experimental images; the experimental images are segmented; nematode identification is performed on the segmented images, thereby completing automatic image processing and automatic quantification of the number of nematodes; a tropism index is automatically calculated according to the number of nematodes in different areas; and a result is judged according to the nematode tropism index.

Description

Technical Field

[0001] The present invention relates to the technical field of biological experiments and image data processing, in particular to a method for automatically processing the trend experiment and image data of a model organism nematode. Background Art

[0002] Caenorhabditis elegans, also known as nematodes, is a widely used model animal. It is small, about 1 mm long, and easy to culture. C. elegans, when cultured in a laboratory at 20°C, has an average lifespan of about two to three weeks, with a developmental period of about three days. C. elegans is also used as a model organism to study developmental and neuroscience issues. Many high-throughput screening experiments using nematodes utilize image acquisition and image processing. The image data generated in high-throughput screening experiments far exceeds the capabilities of manual inspection and analysis, forcing researchers to rely on image processing tools. The purpose of image processing in biological experiments is to utilize advanced computer science techniques and analytical methods to process large amounts of biological image data, thereby helping to solve biological problems.

[0003] The tropism test examines the effects of different chemicals on the sensory behavior of nematodes. It can broadly reflect the neurotoxicity of chemicals and their effects on physiological functions regulated by the nervous system, such as feeding behavior and basic locomotion. The tropism test is a common method for testing chemical toxicity using the nematode model. However, in current nematode tropism tests, there is no automated method for quantifying the tropism index. Conventional manual counting methods are not only time-consuming and labor-intensive, but also subject to biased results due to differences in judgment criteria between experimenters.

[0004] Therefore, how to realize the automatic processing of image data from the nematode tendency experiment is a technical problem that needs to be solved urgently. Summary of the Invention

[0005] The technical task of the present invention is to provide a method for automatic processing of image data of a model organism nematode tendency experiment, so as to solve the problem of how to realize automatic processing of image data of a model organism nematode tendency experiment.

[0006] The technical task of the present invention is achieved in the following manner: a method for automatically processing image data and a trait-oriented experiment of a model organism nematode, the method being specifically as follows:

[0007] Different areas were divided on the NGM culture medium of the model organism C. elegans and volatile substances were added to the designated areas;

[0008] 50-100 young adult nematodes treated with chemicals are spread in the center of the assay plate. Among them, nematode larvae are divided into four stages: L1-L4. L4 is the last stage of nematode larvae, and the adult nematodes at this stage are used for the experiment.

[0009] After being placed in the dark indoors for 0.5-1.5 hours, experimental images were taken under a stereo microscope. The experimental images were used to automatically identify and analyze the number of nematodes in each quadrant;

[0010] Identify the experimental area that actually needs to be analyzed from the experimental image;

[0011] After smoothing, denoising, and removing uneven background brightness in different areas of the experimental image, image segmentation is performed;

[0012] Perform nematode identification on the segmented image, thereby completing automatic image processing and automatic quantification of the number of nematodes;

[0013] Automatically calculate the trend index based on the number of nematodes in different areas;

[0014] The results were judged based on the nematode tropism index.

[0015] Preferably, different regions are divided on the NGM culture medium of the model organism nematode as follows:

[0016] Draw a circle with a diameter of 1 cm around the center of the culture medium;

[0017] The entire culture medium was divided into four quadrants, and a point 1.5 cm away from the center of the culture medium was drawn in each quadrant. The two opposite quadrants were marked with T and C, respectively. That is, the two opposite quadrants were represented by T1 and T2, and the other two opposite quadrants were represented by C1 and C2. Volatile substances were added to the T1 and T2 quadrants; solvent control water was added to the C1 and C2 quadrants.

[0018] Preferably, when automatically identifying and analyzing the number of nematodes in each quadrant, nematodes on the crosshairs and within a 1 cm circle near the center are not counted.

[0019] More preferably, the stereo microscope is OLYMPUS MVX 10; the stereo microscope OLYMPUS MVX 10 includes cameras OLYMPUS DDP73 and MVX-TV0.63XC and a lens OLYMPUS MV PLALO 1X.

[0020] More preferably, the experimental image is an image taken along a crosshair, one experimental image is taken in each quadrant, the experimental image is a 24-bit RGB image, and the pixels of the experimental image are 800*600.

[0021] More preferably, the experimental area that actually needs to be analyzed is identified from the experimental image as follows:

[0022] Identify the right square area in each image;

[0023] The fan-shaped area in the lower right corner was identified within the square area, so that the nematodes in different areas could be counted separately.

[0024] More preferably, nematode identification is performed on the segmented image, thereby completing automatic image processing and automatic quantification of the number of nematodes as follows:

[0025] The actual nematodes in the foreground objects obtained after image segmentation are identified by the size, shape, length, width and aspect ratio of the foreground objects;

[0026] During the experiment, the fan-shaped area in the lower right corner of each image is the area within the 1 cm circle near the center defined previously. The nematodes inside the fan-shaped area and the nematodes in other experimental areas outside the fan-shaped area are automatically identified and counted separately.

[0027] More preferably, the formula for the trend index is as follows:

[0028] CI=[(T1+T2)-(C1+C2)] / [T1+T2+C1+C2];

[0029] Wherein, CI represents the trend index; T1, T2, C1 and C2 represent the number of nematodes identified in four different quadrants, respectively.

[0030] Preferably, the results are judged based on the nematode tropism index as follows:

[0031] When CI>0, it means that the nematodes have a tendency toward the sample;

[0032] When CI < 0, it means that the nematodes have no tendency to the sample;

[0033] When CI = 0, it indicates no behavioral preference.

[0034] The method for the nematode trending experiment and automatic image data processing of the present invention has the following advantages:

[0035] (1) The present invention divides the NGM culture medium of the model organism nematode into different areas and adds volatile substances that are attractive to the nematodes to some of the areas. The nematodes treated with the chemicals are then placed in the prepared designated areas of the culture medium. The freely moving nematodes in the culture medium are observed and experimental images are captured. The experimental images are automatically processed to obtain the number of nematodes residing in different chemical areas, thereby analyzing the effects of different chemicals on the nematode tropism index.

[0036] (2) The present invention realizes automatic and efficient processing of nematode trend experiment image data through nematode trend experiment design and image acquisition and processing, thereby avoiding time-consuming and labor-intensive work and improving the accuracy of nematode trend experiment results;

[0037] (3) The present invention identifies the experimental area that actually needs to be analyzed from the experimental image and counts the nematodes. The nematodes crawl from the center of the fan-shaped area toward the marked point where the volatile substance is dripped, and stay near the marked point. A square area demarcated by a crosshair as the boundary and only counting the nematodes within the square area ensures standardization of the count. Separately counting the nematodes within the fan-shaped area can further identify nematodes that have died due to chemical toxicity or have impaired motility and are unable to crawl.

[0038] (4) The present invention can automatically identify nematodes and quantify the number of nematodes, which can replace the manual technology of the experimenter, not only improving work efficiency and reducing the workload of the experimenter, but also reducing the errors caused by human operation and counting. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present invention will be further described below with reference to the accompanying drawings.

[0040] Attachment Figure 1 This is a schematic diagram of the division of different areas in the nematode culture dish;

[0041] Attachment Figure 2 Schematic diagram of the experimental image;

[0042] Attachment Figure 3 A schematic diagram showing the results of automatically identifying different regions in the captured experimental images;

[0043] Attachment Figure 4 Schematic diagram of the results of identifying nematodes from experimental images;

[0044] Attachment Figure 5 Schematic diagram comparing the results of automatic counting of the present invention and manual counting by an experimenter. DETAILED DESCRIPTION

[0045] The method for the directional experiment of the model organism nematode and the automatic processing of image data of the present invention is described in detail below with reference to the accompanying drawings and specific examples.

[0046] Example:

[0047] This embodiment provides a method for a nematode tropism experiment and automatic image data processing for a model organism. The method involves dividing a NGM culture medium of the model organism nematode into different areas and adding volatile substances that are attractive to the nematodes to some of the areas. The chemically treated nematodes are then placed in the prepared designated areas of the culture medium. The freely moving nematodes in the culture medium are observed and experimental images are captured. The experimental images are automatically processed to obtain the number of nematodes residing in different chemical areas, thereby analyzing the effects of different chemicals on the nematode tropism index. The details are as follows:

[0048] S1. Divide the NGM culture medium of the model organism C. elegans into different areas and drip volatile substances into the designated areas; the areas are divided as follows:

[0049] S101. Draw a circle with a diameter of 1 cm around the center of the culture medium;

[0050] S102, as attached Figure 1 As shown, the entire culture medium is evenly divided into 4 quadrants, and a point 1.5 cm away from the center of the culture medium is drawn in each quadrant. The two quadrants opposite to each other are marked with T and C, that is, the two opposite quadrants are represented by T1 and T2, and the other two opposite quadrants are represented by C1 and C2. Volatile substances are added to quadrants T1 and T2; solvent control water is added to quadrants C1 and C2.

[0051] S2. Spread 50-100 young adult nematodes treated with chemicals in the center of the assay plate. Among them, nematode larvae are divided into four stages, L1-L4, and L4 is the last stage of nematode larvae. Adult nematodes at the L4 stage are used for the experiment. After being placed in the dark indoors for 1 hour, experimental images are taken under a stereo microscope. The experimental images are used to automatically identify and analyze the number of nematodes in each quadrant. Among them, when automatically identifying and analyzing the number of nematodes in each quadrant, the nematodes on the crosshairs and within the 1 cm circle near the center are not counted. Under a stereo microscope (OLYMPUS MVX 10) [camera (OLYMPUS DP73, MVX-TV0.63XC) and lens (OLYMPUS MV PLALO 1X)], take experimental images along the crosshairs, as shown in the attached figure. Figure 2 As shown, an experimental image is taken in each quadrant. The experimental image is a 24-bit RGB image with a pixel size of 800*600.

[0052] S3. Identify the experimental area that actually needs to be analyzed from the experimental image; the details are as follows:

[0053] S301, identifying the right square area in each image;

[0054] S302: Identify the fan-shaped area in the lower right corner of the square area, so as to count the nematodes in different areas separately.

[0055] As attached Figure 3 As shown in the figure, the results of automatically identifying different areas in the captured experimental image are shown, where the black and white areas correspond to the attached Figure 2 The right square area and the white area in the lower right corner correspond to the attached Figure 2 This counting method also offers the following advantages: nematodes crawl from the center of the fan-shaped area toward the marked point where the volatile substance has been dripped, and then settle near the marked point. Counting only nematodes within a square area defined by a crosshair ensures standardization. Counting only nematodes within the fan-shaped area can also identify nematodes that have died from chemical toxicity or have impaired motility and are unable to crawl.

[0056] S4. After smoothing, denoising, and removing uneven background brightness in different areas of the experimental image, image segmentation is performed;

[0057] S5. Identify nematodes on the segmented image, thereby completing automatic image processing and automatic quantification of the number of nematodes; the details are as follows:

[0058] S501, identifying the actual nematodes in the foreground objects obtained after image segmentation by using the features of size, shape, length, width and aspect ratio of the foreground objects;

[0059] S502. During the experiment, the fan-shaped area in the lower right corner of each image is the area within the 1 cm circle near the center defined above. The nematodes within the fan-shaped area and the nematodes in other experimental areas excluding the fan-shaped area are automatically identified and counted separately.

[0060] As attached Figure 4 As shown in the figure, the results of identifying nematodes from the experimental images are shown in the figure. Figure 4 Nematodes identified in the Figure 3 The number of nematodes in different areas can be obtained by identifying the areas in the figure. Figure 5 As shown in the figure, the results of automatic counting and manual counting by the experimenter are compared. Figure 5 It can be seen that the results of automatic identification and quantification of nematodes are very close to those of manual counting, achieving a relatively good effect of automatic identification and quantification of nematodes, thus proving the effectiveness of our method, and that it can replace manual counting by experimenters, improve work efficiency, and reduce errors in human operation and counting.

[0061] S6. Automatically calculate the trend index based on the number of nematodes in different areas. The trend index formula is as follows:

[0062] CI=[(T1+T2)-(C1+C2)] / [T1+T2+C1+C2];

[0063] Wherein, CI represents the trend index; T1, T2, C1 and C2 represent the number of nematodes identified in four different quadrants, respectively.

[0064] S7. Determine the results based on the nematode tropism index; details are as follows:

[0065] When CI>0, it means that the nematodes have a tendency toward the sample;

[0066] When CI < 0, it means that the nematodes have no tendency to the sample;

[0067] When CI = 0, it indicates no behavioral preference.

[0068] In actual experiments, in order to ensure the reliability of the results, multiple sets of technical repetitions should be designed for each sample.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for automatic processing of nematode trending experiments and image data, characterized in that: The method is as follows: Different areas were divided on the NGM culture medium of the model organism C. elegans and volatile substances were added to the designated areas; 50-100 chemically treated young adult nematodes were spread in the center of the assay plate; After being placed in the dark indoors for 0.5-1.5 hours, experimental images were taken under a stereo microscope. The experimental images were used to automatically identify and analyze the number of nematodes in each quadrant; Identify the experimental area that actually needs to be analyzed from the experimental image; After smoothing, denoising, and removing uneven background brightness in different areas of the experimental image, image segmentation is performed; Perform nematode identification on the segmented image, thereby completing automatic image processing and automatic quantification of the number of nematodes; Automatically calculate the trend index based on the number of nematodes in different areas; The results were judged based on the nematode tropism index; Among them, the different areas are divided on the NGM culture medium of the model organism nematode as follows: Draw a circle with a diameter of 1 cm around the center of the culture medium; The entire culture medium was divided into four equal quadrants. A point 1.5 cm from the center of the culture medium was drawn in each quadrant. Two opposing quadrants were marked with T and C, respectively. That is, the two opposing quadrants were designated T1 and T2, and the other two opposing quadrants were designated C1 and C2. Volatile substances were added dropwise to quadrants T1 and T2; solvent control water was added dropwise to quadrants C1 and C2. When automatically identifying and analyzing the number of nematodes in each quadrant, nematodes on the crosshairs and within a 1-cm circle near the center were not counted; The experimental areas that need to be analyzed are identified from the experimental images as follows: Identify the right square area in each image; Identify the fan-shaped area in the lower right corner of the square area, so that the nematodes in different areas can be counted separately; The segmented image is used to identify nematodes, thereby completing the automatic processing of the image and the automatic quantification of the number of nematodes as follows: The actual nematodes in the foreground objects obtained after image segmentation are identified by the size, shape, length, width and aspect ratio of the foreground objects; During the experiment, the fan-shaped area in the lower right corner of each image is the area within the 1 cm circle near the center defined above. The nematodes inside the fan-shaped area and the nematodes in other experimental areas outside the fan-shaped area are automatically identified and counted separately.

2. The method for the nematode trending experiment and automatic image data processing according to claim 1, characterized in that: The stereo microscope used is OLYMPUS MVX 10; the stereo microscope OLYMPUS MVX 10 includes cameras OLYMPUS DP73 and MVX-TV0.63XC and a lens OLYMPUS MV PLALO 1X.

3. The method for the nematode trending experiment and automatic image data processing according to claim 1, characterized in that: The experimental image is an image taken along the crosshairs, one experimental image is taken in each quadrant, the experimental image is a 24-bit RGB image, and the pixel size of the experimental image is 800*600.

4. The method for the nematode trending experiment and automatic image data processing according to claim 1, characterized in that: The formula for the trend index is as follows: CI=[(T1+T2)-(C1+C2)] / [T1+T2+C1+C2]; Wherein, CI represents the trend index; T1, T2, C1 and C2 represent the number of nematodes identified in four different quadrants, respectively.

5. The method for the nematode trending experiment and automatic image data processing according to claim 1, characterized in that: The results are judged according to the nematode tropism index as follows: When CI>0, it means that the nematodes have a tendency toward the sample; When CI < 0, it means that the nematodes have no tendency to the sample; When CI = 0, it indicates no behavioral preference.

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