CN107084984A - A method for image measurement of muscle fiber cell parameters - Google Patents

Abstract

The invention discloses a method for image measurement of muscle fiber cell parameters. In the invention, after preparing the muscle fiber sample of pork, we collect microscopic images, and then use image processing algorithms to write programs to automatically segment muscle fiber cells. And on this basis, the calculation of parameters such as the area and diameter of a single cell is completed, so as to realize the automatic measurement of pork muscle fiber parameters, reduce the workload of measurement, and improve the accuracy of measurement.

Description

A method for image measurement of muscle fiber cell parameters

technical field

The invention belongs to the detection field, and in particular relates to an image measurement method for muscle fiber cell parameters.

Background technique

Quantitative analysis of muscle fibers can be used to evaluate the influence of different feeding or growth environments on muscles, and can also be used for selection of species. The existing analysis methods basically still rely on manual measurement, that is, to observe the muscle samples under the microscope by humans and perform corresponding measurements. There are problems such as heavy labor, low work efficiency, and poor accuracy. Some literatures use image processing software to help measure, but still rely on manual selection, and there is no major improvement in terms of labor and work efficiency.

For example, in the paper "Quantitative calculation of the number and area of muscle fiber types by different image analysis software", the author used image processing software such as ImageJ, Image Pro plus and Photoshop to manually select muscle fiber cells through manual interaction, and then the image processing software calculated its area. Although the use of image processing software to calculate the area improves the accuracy of measurement and avoids errors caused by humans in the past when estimating the area of irregularly shaped muscle fiber cells, the measurement process still relies on manual labor in essence.

In the present invention, after preparing the muscle fiber sample of pork, we collect the microscopic image, and then use the image processing algorithm to write a program to automatically segment out the muscle fiber cells, and complete the parameters such as the area and diameter of a single cell on this basis. The calculation can realize the automatic measurement of pork muscle fiber parameters, reduce the workload of measurement and improve the accuracy of measurement.

Contents of the invention

The object of the present invention is to provide a kind of image measurement method of muscle fiber cell parameter, the present invention can finish the measurement of pork muscle fiber parameter automatically by the method for image processing, and labor load is small, and work efficiency is high, and result is stable, and accuracy is high, and It can avoid erroneous results introduced by fatigue or supervisor's judgment factors in manual operation.

To achieve the above object, the technical solution of the present invention is as follows:

A kind of image measurement method of muscle fiber cell parameter, comprises the steps:

(1) obtain the image of the muscle fiber and the ruler of the image;

(2) Image measurement of muscle fiber parameters:

(2.1) Convert the color image I obtained by shooting into a grayscale image G, and then perform threshold segmentation on G to obtain _a binary image B1 _; in B1, the dark muscle fiber cell area is black, and the background area is white;

(2.2) Carry out connected domain marking to B ₁ , then remove the white area with an area smaller than a given threshold T _A1 ; obtain the inverse color image of the binary image after the white area is removed, and obtain B ₂ ;

(2.3) Carry out connected domain marking on B ₂ to obtain the marked image L ₁ , and then remove the border area in L ₁ to obtain the marked image L ₂ ;

(2.4) remove the region in L2 whose area is smaller _than a given threshold T _A2 or larger than a given threshold T _A3 , to obtain a marked image L3 _;

(2.5) For each region in L3, calculate the ratio r _of its area to the area of the convex hull, remove the region whose r value is less _than a given threshold T _acar in L3, and obtain the marked image L4 _;

(2.6) Carry out hole filling for each area in L ₄ to obtain the muscle fiber area;

(2.7) Calculate the muscle fiber cell parameters according to the muscle fiber area.

As a further improvement, in step (2.7), the muscle fiber cell parameters include the actual area of the muscle fiber, and the actual area calculation method of the muscle fiber is as follows:

Calculate the relative area of the muscle fiber region, that is, the number of pixels A _I in the muscle fiber region; calculate the actual length s corresponding to the length of 1 pixel in the image according to the scale given in the image, so the actual area of the muscle fiber A=A _I ·s ² .

As a further improvement, in step (2.7), the muscle fiber cell parameters include the actual diameter of the muscle fiber, and the actual diameter calculation method of the muscle fiber is as follows:

Extract the contour of the muscle fiber area, calculate the distance between each pair of different points on the contour, and take the maximum value of the distance between all pairs of different points as the diameter d _I of the muscle fiber, and the unit is pixel; according to the scale given in the image, calculate The actual length s corresponding to the length of 1 pixel in the image, so the actual diameter of the muscle fiber d=d _I ·s.

As a further improvement, the border area in step (2.3) means that if at least one pixel point in a certain muscle fiber cell area falls on the outer edge of the image, that is, outside the top, bottom row, leftmost column or rightmost column of the image area, the area _of the muscle fiber cells in the image L1 is called the welt area.

As a further improvement, the muscle fiber cells are pork fiber cells.

As a further improvement, in step (1), the image of the muscle fiber is obtained by taking microscopic images of the sample in multiple fields of view by the camera.

As a further improvement, the sample is prepared through the following steps:

(1.1) Fix: fix the sample with 4% paraformaldehyde for 24 hours;

(1.2) Dehydration: Put the washed samples into 70%, 80%, 90%, 100%, and 100% alcohol and soak them overnight, 8 hours, 8 hours, 1 hour, and 1 hour to remove moisture in the sample;

(1.3) Transparent: Soak in 1:1 xylene: absolute alcohol for 1 hour, then soak in xylene for 1 hour to replace the absolute alcohol in the tissue block;

(1.4) Wax immersion and embedding: put the transparent sample into 1:1 xylene:paraffin for transition for 30 minutes, then put it into a wax cup filled with paraffin for 30 minutes, and put it in an incubator. After the paraffin is melted, the temperature of the incubator should not be higher than 56°C. Put the tissue block soaked in wax into a carton filled with melted paraffin, so that the tissue block is embedded in paraffin, and quickly placed in a water basin. Ready to use after cooling;

(1.5) Slicing: the prepared tissue wax blocks were trimmed and adhered to the platform wood, and then cut into 6 μm thick slices with a microtome, and the slices were continuously formed into wax strips;

(1.6) Spreading and sticking slices: Spread the cut wax strips in warm water at 30°C, and then use 1:1 egg white: glycerin mixture as the adhesive to stick the tissue slices on the glass slides. Then place it in a 37°C incubator and dry it for no less than 24 hours;

(1.7) Dewaxing and rehydration: Adjust the temperature of the water bath to 60°C. When the water temperature is controlled at 60°C, put the slices together with the slice rack into a dry staining vat, put them in the water bath, and cover them for 30 minutes. until the wax melts;

(1.8) Paraffin sections were dewaxed with xylene I and II for 5 minutes each, and then placed in alcohol solutions of 100%, 95%, 90%, 80%, and 70% concentrations for 3-5 minutes each, and then put into distilled water 3min;

(1.9) Staining: Put the slices into hematoxylin and stain for 15 minutes;

(1.10) Washing: Rinse with running water for 15 minutes; make the slices turn blue;

(1.11) Differentiation: Put the slices into 1% hydrochloric acid ethanol solution to fade the color for 2 seconds to tens of seconds; see that the slices turn red, and the color is lighter;

(1.12) Rinsing: put the slices into running water to make them turn blue again;

(1.13) Dehydration Ⅰ: slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 minutes each;

(1.14) Counterstaining: contrast staining with 0.5% eosin ethanol solution for 1 min;

(1.15) Dehydration II: Put the slices into 95% ethanol to wash away the excess red color, then put them into absolute ethanol for 3-5 minutes; finally, absorb the excess ethanol with absorbent paper;

(1.16) Transparency: Put the slices into xylene I and II for 3-5 minutes each;

(1.17) Sealing: neutral gum sealing.

detailed description

Example

The method proposed in the present invention includes two main stages: the preparation and image acquisition stage of the pork muscle sample, and the image processing and measurement stage.

(1) Sample preparation and image acquisition

(1.1) Fixing: Fix the sample with 4% paraformaldehyde for 24 hours.

(1.2) Dehydration: Put the washed samples into alcohols of 70%, 80%, 90%, 100% (absolute alcohol), and 100% (absolute alcohol) through overnight, 8 hours, and 8 hours respectively , 1 hour, and 1 hour soaking to remove the moisture in the sample.

(1.3) Transparent: Soak in 1:1 xylene:absolute alcohol for 1 hour, and then soak in xylene for 1 hour to replace the absolute alcohol in the tissue block.

(1.4) Wax immersion and embedding: Put the transparent sample into 1:1 xylene: paraffin for transition for 30 minutes, then put it into a wax cup filled with paraffin for 30 minutes (need to put it in an incubator) After the paraffin is melted, the temperature of the incubator should not be higher than 56°C). Put the wax-soaked tissue block into a carton filled with melted paraffin, embed the tissue block in paraffin, put it in the water basin quickly, and wait for it to cool down for later use.

(1.5) Slicing: After trimming and cutting the prepared tissue wax block, stick it to the platform wood and cut it into 6 μm thick slices with a microtome. The slices should be continuous into wax strips.

(1.6) Spreading and sticking slices: Spread the cut wax strips in warm water at 30°C, and then use 1:1 egg white: glycerin mixture as the adhesive to stick the tissue slices on the glass slides. Then place it in a 37°C incubator and dry it (not less than 24h).

(1.7) Dewaxing and rehydration: Adjust the temperature of the water bath to 60°C. When the water temperature is controlled at 60°C, put the slices together with the slice rack into a dry staining vat, put them in the water bath, and cover the lid (optional). Seal), 30min until the wax melts.

(1.8) Paraffin sections were dewaxed with xylene I and II for 5 minutes each, and then placed in alcohol solutions of 100%, 95%, 90%, 80%, and 70% concentrations for 3-5 minutes each, and then put into distilled water 3min.

(1.9) Staining: Put the slices into hematoxylin and stain for about 15 minutes.

(1.10) Washing: Rinse with running water for about 15 minutes. Make the slices turn blue (or put them in alkaline water), but be careful not to run too much water to prevent the slices from falling off.

(1.11) Differentiation: put the slice into 1% hydrochloric acid ethanol solution to fade the color, about 2 seconds to tens of seconds. See that the slices turn red, and the color is lighter.

(1.12) Rinsing: Put the slices into running water to restore the blue color.

(1.13) Dehydration Ⅰ: slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 minutes each.

(1.14) Counterstaining: contrast staining with 0.5% eosin ethanol solution for 1 min.

(1.15) Dehydration II: Put the slices into 95% ethanol to wash off excess red, and then put them into absolute ethanol for 3-5 minutes. Finally, blot the excess ethanol with absorbent paper.

(1.16) Transparency: Place the slices in xylene I and II for 3-5 minutes each.

(1.17) Sealing: neutral gum sealing

(1.18) Acquire images: place the sample under the microscope, and take microscopic images of the sample with multiple fields of view through the camera, and a ruler should be added to the image.

(2) Image measurement of muscle fiber parameters

(2.1) Transform the captured color image I into a grayscale image G, and then perform threshold segmentation on G to obtain a binary image B ₁ . In B ₁ , the dark muscle fiber cell area is black, and the background area is white;

(2.2) Carry out connected domain marking _on B1, and then remove those white areas whose area is smaller than a given threshold T _A1 . Calculate the inverse color image of the binary image after region removal to obtain B ₂ ;

(2.3) Mark the connected domain of B ₂ to obtain the marked image L ₁ , and then remove the border area in L ₁ , that is, at least one pixel in the area falls on the outer edge of the image (that is, the uppermost and lowermost rows of the image and the leftmost and rightmost columns) to get the marked image L ₂ ; the border area means that individual muscle fiber cells are not all within the range of image L ₁ , but part of them are inside image L ₁ and the other part is outside image L ₁ , the part _of the incomplete muscle fiber cell area in the image L1 is the so-called welt area.

(2.4) remove the region in L2 whose area is smaller _than a given threshold T _A2 or larger than a given threshold T _A3 , to obtain a marked image L3 _;

(2.5) For each region in L3, calculate the ratio r _of its area to the area of the convex hull, remove the region whose r value is less _than a given threshold T _acar in L3, and obtain the marked image L4 _;

(2.6) For each area in L ₄ , it is filled with holes to obtain the muscle fiber area of pork;

(2.7) calculate the area (i.e. the number of pixels) A _I of the muscle fiber region;

(2.8) Extract the contour of the muscle fiber region, calculate the distance between every pair of different points on the contour, and take the maximum value of the distance of all these point pairs as the diameter (in pixels) d _I of the muscle fiber;

(2.9) According to the scale given in the image, calculate the actual length s corresponding to the length of 1 pixel in the image, so the actual area of the muscle fiber is A=A _I ·s ² , and the actual diameter is d=d _I ·s.

Although the present invention is proposed for the measurement of muscle fiber parameters of pork, if it is the measurement of muscle fiber parameters of other meats, if the muscle fiber morphology of these meats is similar to that of pork, then the method of the present invention is also applicable, so it should be regarded as the present invention the scope covered.

The muscle fiber parameters referred to in the present invention mainly refer to the measurement of the area and diameter of the muscle fiber cross section.

As mentioned above, the implementation examples of the present invention have been described in detail, but as long as they do not depart from the characteristics and effects of the present invention, many modifications can be made, which will be obvious to those skilled in the art. Therefore, these Modified examples are also all included within the protection scope of the present invention.

Claims (7)

1. an image measurement method of muscle fiber cell parameter, is characterized in that, comprises the steps: (1) obtain the image of the muscle fiber and the ruler of the image; (2) Image measurement of muscle fiber parameters: (2.1) Convert the color image I obtained by shooting into a grayscale image G, and then perform threshold segmentation on G to obtain _a binary image B1 _; in B1, the dark muscle fiber cell area is black, and the background area is white; (2.2) Carry out connected domain marking to B ₁ , then remove the white area with an area smaller than a given threshold T _A1 ; obtain the inverse color image of the binary image after the white area is removed, and obtain B ₂ ; (2.3) Carry out connected domain marking on B ₂ to obtain the marked image L ₁ , and then remove the border area in L ₁ to obtain the marked image L ₂ ; (2.4) remove the region in L2 whose area is smaller _than a given threshold T _A2 or larger than a given threshold T _A3 , to obtain a marked image L3 _; (2.5) For each region in L3, calculate the ratio r _of its area to the area of the convex hull, remove the region whose r value is less _than a given threshold T _acar in L3, and obtain the marked image L4 _; (2.6) Carry out hole filling for each area in L ₄ to obtain the muscle fiber area; (2.7) Calculate the muscle fiber cell parameters according to the muscle fiber area. 2. the image measurement method of muscle fiber cell parameter as claimed in claim 1, is characterized in that, in step (2.7), described muscle fiber cell parameter comprises the actual area of muscle fiber, and the actual area calculation method of muscle fiber is as follows: Calculate the relative area of the muscle fiber region, that is, the number of pixels A _I in the muscle fiber region; calculate the actual length s corresponding to the length of 1 pixel in the image according to the scale given in the image, so the actual area of the muscle fiber A=A _I ·s ² . 3. the image measurement method of muscle fiber cell parameter as claimed in claim 1, is characterized in that, in step (2.7), described muscle fiber cell parameter comprises the actual diameter of muscle fiber, and the actual diameter calculation method of muscle fiber is as follows: Extract the contour of the muscle fiber area, calculate the distance between each pair of different points on the contour, and take the maximum value of the distance between all pairs of different points as the diameter d _I of the muscle fiber, and the unit is pixel; according to the scale given in the image, calculate The actual length s corresponding to the length of 1 pixel in the image, so the actual diameter of the muscle fiber d=d _I ·s. 4. the image measurement method of muscle fiber cell parameter as claimed in claim 1, is characterized in that, if at least one pixel in a certain muscle fiber cell region falls on the outer edge of image in the welt area in step (2.3) , that is, the area outside the uppermost, lowermost row, leftmost column or rightmost column of the image, then the area _of the muscle fiber cells in the image L1 is called the border area. 5. The image measurement method of muscle fiber cell parameter as claimed in claim 1, is characterized in that, described muscle fiber cell is pork fiber cell. 6. The image measurement method of muscle fiber cell parameters as claimed in claim 1, characterized in that, in step (1), the image of the muscle fiber is obtained by taking a sample microscopic image of multiple fields of view by a camera. 7. the image measurement method of muscle fiber cell parameter as claimed in claim 6, is characterized in that, described sample is made through the following steps: (1.1) Fix: fix the sample with 4% paraformaldehyde for 24 hours; (1.2) Dehydration: Put the washed samples into 70%, 80%, 90%, 100%, and 100% alcohol and soak them overnight, 8 hours, 8 hours, 1 hour, and 1 hour to remove moisture in the sample; (1.3) Transparent: Soak in 1:1 xylene: absolute alcohol for 1 hour, then soak in xylene for 1 hour to replace the absolute alcohol in the tissue block; (1.4) Wax immersion and embedding: put the transparent sample into 1:1 xylene:paraffin for transition for 30 minutes, then put it into a wax cup filled with paraffin for 30 minutes, and put it in an incubator. After the paraffin is melted, the temperature of the incubator should not be higher than 56°C. Put the tissue block soaked in wax into a carton filled with melted paraffin, so that the tissue block is embedded in paraffin, and quickly placed in a water basin. Ready to use after cooling; (1.5) Slicing: the prepared tissue wax blocks were trimmed and adhered to the platform wood, and then cut into 6 μm thick slices with a microtome, and the slices were continuously formed into wax strips; (1.6) Spreading and sticking slices: Spread the cut wax strips in warm water at 30°C, and then use 1:1 egg white: glycerin mixture as the adhesive to stick the tissue slices on the glass slides. Then place it in a 37°C incubator and dry it for no less than 24 hours; (1.7) Dewaxing and rehydration: Adjust the temperature of the water bath to 60°C. When the water temperature is controlled at 60°C, put the slices together with the slice rack into a dry staining vat, put them in the water bath, and cover them for 30 minutes. until the wax melts; (1.8) Paraffin sections were dewaxed with xylene I and II for 5 minutes each, and then placed in alcohol solutions of 100%, 95%, 90%, 80%, and 70% concentrations for 3-5 minutes each, and then put into distilled water 3min; (1.9) Staining: Put the slices into hematoxylin and stain for 15 minutes; (1.10) Washing: Rinse with running water for 15 minutes; make the slices turn blue; (1.11) Differentiation: Put the slices into 1% hydrochloric acid ethanol solution to fade the color for 2 seconds to tens of seconds; see that the slices turn red, and the color is lighter; (1.12) Rinsing: put the slices into running water to make them turn blue again; (1.13) Dehydration Ⅰ: slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 minutes each; (1.14) Counterstaining: contrast staining with 0.5% eosin ethanol solution for 1 min; (1.15) Dehydration Ⅱ: Put the slices into 95% ethanol to wash away the excess red color, then put them into absolute ethanol for 3-5 minutes; finally absorb the excess ethanol with absorbent paper; (1.16) Transparency: Put the slices into xylene I and II for 3-5 minutes each; (1.17) Sealing: neutral gum sealing.