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

A method for image measurement of muscle fiber cell parameters Download PDF

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CN107084984A
CN107084984A CN201710252244.2A CN201710252244A CN107084984A CN 107084984 A CN107084984 A CN 107084984A CN 201710252244 A CN201710252244 A CN 201710252244A CN 107084984 A CN107084984 A CN 107084984A
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muscle fiber
image
area
slices
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郭斯羽
朱辉
鲍美华
凌志刚
刘敏
温和
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Hunan University
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • GPHYSICS
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    • GPHYSICS
    • G06COMPUTING; CALCULATING OR COUNTING
    • G06TIMAGE DATA PROCESSING OR GENERATION, IN GENERAL
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    • G06TIMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00Indexing scheme for image analysis or image enhancement
    • G06T2207/10Image acquisition modality
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Abstract

本发明公开了一种肌纤维细胞参数的图像测量方法,在本发明中,我们在制备了猪肉的肌纤维样本之后,采集显微图像,然后通过图像处理算法,编写程序来自动地分割出肌纤维细胞,并在此基础上完成单个细胞的面积、直径等参数的计算,从而实现猪肉肌纤维参数的自动测量,减少测量的工作量,提高测量的准确度。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

一种肌纤维细胞参数的图像测量方法A method for image measurement of muscle fiber cell parameters

技术领域technical field

本发明属于检测领域,尤其涉及一种肌纤维细胞参数的图像测量方法。The invention belongs to the detection field, and in particular relates to an image measurement method for muscle fiber cell parameters.

背景技术Background technique

对肌肉纤维的定量分析可用于评价不同的饲养或成长环境对于肌肉的影响,也可用于选种等工作。现有的分析方法基本仍然依赖于人工测量,即通过人来观察显微镜下的肌肉样本,并进行相应的测量,存在着劳动量大、工作效率低、准确性差等问题。有部分文献中使用了图像处理软件来帮助测量,但仍然依赖于人工选择,劳动量和工作效率等方面没有大的改观。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.

例如在论文《不同图像分析软件定量计算肌纤维类型数量及面积》中,作者使用了ImageJ、Image Pro plus和Photoshop等图像处理软件,通过人工交互的方式,手工选择肌纤维细胞,然后由图像处理软件计算其面积。虽然利用图像处理软件计算面积提高了测量的准确性,避免了过去由人估算不规则形状的肌纤维细胞的面积时造成的误差,但是测量过程本质上仍然依赖于人工。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.

在本发明中,我们在制备了猪肉的肌纤维样本之后,采集显微图像,然后通过图像处理算法,编写程序来自动地分割出肌纤维细胞,并在此基础上完成单个细胞的面积、直径等参数的计算,从而实现猪肉肌纤维参数的自动测量,减少测量的工作量,提高测量的准确度。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.

发明内容Contents of the invention

本发明的目的在于提供一种肌纤维细胞参数的图像测量方法,本发明通过图像处理的方法,能够自动地完成猪肉肌纤维参数的测量,劳动量小,工作效率高,结果稳定,准确度高,并可避免人工操作中因疲劳或主管判断因素所引入的错误结果。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.

为实现上述目的,本发明的技术方案如下所示:To achieve the above object, the technical solution of the present invention is as follows:

一种肌纤维细胞参数的图像测量方法,包括如下步骤:A kind of image measurement method of muscle fiber cell parameter, comprises the steps:

(1)获得肌纤维的图像和图像的标尺;(1) obtain the image of the muscle fiber and the ruler of the image;

(2)肌纤维参数的图像测量:(2) Image measurement of muscle fiber parameters:

(2.1)将拍摄得到的彩色图像I转化为灰度图像G,然后对G进行阈值分割得到二值图像B1;B1中,深色的肌纤维细胞区域为黑色,背景区域为白色;(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)对B1进行连通域标记,然后去除面积小于给定阈值TA1的白色区域;求取白色区域去除之后的二值图像的反色图像,得到B2(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)对B2进行连通域标记,得到标记图像L1,然后去除L1中的贴边区域,得到标记图像L2(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)去除L2中面积小于给定阈值TA2或大于给定阈值TA3的区域,得到标记图像L3(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)对L3中的每个区域,计算其面积与凸包面积之比r,去除L3中r值小于给定阈值Tacar的区域,得到标记图像L4(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)对L4中的每个区域,进行孔洞填充,即得肌纤维区域;(2.6) Carry out hole filling for each area in L 4 to obtain the muscle fiber area;

(2.7)根据肌纤维区域计算得到肌纤维细胞参数。(2.7) Calculate the muscle fiber cell parameters according to the muscle fiber area.

进一步的改进,步骤(2.7)中,所述肌纤维细胞参数包括肌纤维的实际面积,肌纤维的实际面积计算方法如下: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:

计算肌纤维区域的相对面积即肌纤维区域内的像素点个数AI;根据图像中所给的标尺,计算图像中1像素长度所对应的实际长度s,于是肌纤维的实际面积A=AI·s2Calculate 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 .

进一步的改进,步骤(2.7)中,所述肌纤维细胞参数包括肌纤维的实际直径,肌纤维的实际直径计算方法如下: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:

提取肌纤维区域的轮廓,计算轮廓上每对相异点之间的距离,以所有相异点对的距离的最大值为肌纤维的直径dI,单位为像素;据图像中所给的标尺,计算图像中1像素长度所对应的实际长度s,于是肌纤维的实际直径d=dI·s。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.

进一步的改进,步骤(2.3)中的贴边区域即若某个肌纤维细胞区域中至少有一个像素点落在了图像的外缘,即图像的最上、最下行、最左列或最右列外的区域,则此肌纤维细胞在图像L1内的区域称作贴边区域。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.

进一步的改进,所述肌纤维细胞为猪肉纤维细胞。As a further improvement, the muscle fiber cells are pork fiber cells.

进一步的改进,步骤(1)中,肌纤维的图像通过相机拍摄多个视野的样本显微图像获得。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.

进一步的改进,所述样本通过以下步骤制得:As a further improvement, the sample is prepared through the following steps:

(1.1)固定:用4%的多聚甲醛固定样本24小时;(1.1) Fix: fix the sample with 4% paraformaldehyde for 24 hours;

(1.2)脱水:将洗好后的样品放入为70%、80%、90%、100%、100%的酒精中分别经过过夜、8小时、8小时、1小时、1小时浸泡以脱去样品中的水分;(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)透明:以1:1的二甲苯:无水酒精浸泡1小时,再用二甲苯浸泡1小时,以置换出组织块中的无水酒精;(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)浸蜡和包埋:将透明好的样品放入1:1的二甲苯:石蜡中过渡30分钟,再将其放入装有石蜡的蜡杯中30分钟,需在温箱中将石蜡熔化后进行,温箱温度不得高于56℃,将浸好蜡的组织块放入盛有熔化石蜡的纸盒内,使组织块包埋于石蜡中,迅速放在水盆中,待其冷却后备用;(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)切片:将制好的组织蜡块经修切后粘附于台木后用切片机切成6μm厚的薄片,薄片连续成蜡带状;(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)展片和粘片:将切成的蜡带展于30℃的温水中,再以1:1的鸡蛋清:甘油混合剂为粘附剂,将组织薄片粘附于载玻片上,再将其置于37℃温箱中烘干,不少于24h;(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)脱蜡复水:将水浴锅温度调至60℃,待水温控制在60℃时,将切片连同切片架放入一个干燥的染色缸内,放入水浴锅中,盖上盖子,30min至蜡熔化;(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)石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各5min,然后放入100%、95%、90%、80%、70%各级浓度的酒精溶液中各3-5min,再放入蒸馏水中3min;(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)染色:切片放入苏木精中染色15min;(1.9) Staining: Put the slices into hematoxylin and stain for 15 minutes;

(1.10)水洗:用自来水流水冲洗15min;使切片颜色变蓝;(1.10) Washing: Rinse with running water for 15 minutes; make the slices turn blue;

(1.11)分化:将切片放入1%盐酸乙醇液中褪色,2秒至数十秒钟;见切片变红,颜色较浅即可;(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)漂洗:切片再放入自来水流水中使其恢复蓝色;(1.12) Rinsing: put the slices into running water to make them turn blue again;

(1.13)脱水Ⅰ:切片入50%乙醇→70%乙醇→80%乙醇中各3-5min;(1.13) Dehydration Ⅰ: slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 minutes each;

(1.14)复染:用0.5%伊红乙醇液对比染色1min;(1.14) Counterstaining: contrast staining with 0.5% eosin ethanol solution for 1 min;

(1.15)脱水Ⅱ:将切片放入95%乙醇中洗去多余的红色,然后放入无水乙醇中3-5min;最后用吸水纸吸干多余的乙醇;(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)透明:切片放入二甲苯Ⅰ、Ⅱ中各3-5min;(1.16) Transparency: Put the slices into xylene I and II for 3-5 minutes each;

(1.17)封藏:中性树胶封存。(1.17) Sealing: neutral gum sealing.

具体实施方式detailed description

实施例Example

本发明所提方法包括两个主要阶段:猪肉肌肉样本的制备和图像获取阶段,以及图像处理和测量阶段。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)样本制备与图像获取(1) Sample preparation and image acquisition

(1.1)固定:用4%的多聚甲醛固定样本24小时。(1.1) Fixing: Fix the sample with 4% paraformaldehyde for 24 hours.

(1.2)脱水:将洗好后的样品放入为70%、80%、90%、100%(无水酒精)、100%(无水酒精)的酒精中分别经过过夜、8小时、8小时、1小时、1小时浸泡以脱去样品中的水分。(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)透明:以1:1的二甲苯:无水酒精浸泡1小时,再用二甲苯浸泡1小时,以置换出组织块中的无水酒精。(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)浸蜡和包埋:将透明好的样品放入1:1的二甲苯:石蜡中过渡30分钟,再将其放入装有石蜡的蜡杯中30分钟(需在温箱中将石蜡熔化后进行,温箱温度不得高于56℃)。将浸好蜡的组织块放入盛有熔化石蜡的纸盒内,使组织块包埋于石蜡中,迅速放在水盆中,待其冷却后备用。(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)切片:将制好的组织蜡块经修切后粘附于台木后用切片机切成6μm厚的薄片,薄片应该连续成蜡带状。(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)展片和粘片:将切成的蜡带展于30℃的温水中,再以1:1的鸡蛋清:甘油混合剂为粘附剂,将组织薄片粘附于载玻片上,再将其置于37℃温箱中烘干(不少于24h)。(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)脱蜡复水:将水浴锅温度调至60℃,待水温控制在60℃时,将切片连同切片架放入一个干燥的染色缸内,放入水浴锅中,盖上盖子(可密封),30min至蜡熔化。(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)石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各5min,然后放入100%、95%、90%、80%、70%各级浓度的酒精溶液中各3-5min,再放入蒸馏水中3min。(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)染色:切片放入苏木精中染色约15min。(1.9) Staining: Put the slices into hematoxylin and stain for about 15 minutes.

(1.10)水洗:用自来水流水冲洗约15min。使切片颜色变蓝(或放入碱性水中也可),但要注意流水不能过大,以防切片脱落。(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)分化:将切片放入1%盐酸乙醇液中褪色,约2秒至数十秒钟。见切片变红,颜色较浅即可。(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)漂洗:切片再放入自来水流水中使其恢复蓝色。(1.12) Rinsing: Put the slices into running water to restore the blue color.

(1.13)脱水Ⅰ:切片入50%乙醇→70%乙醇→80%乙醇中各3-5min。(1.13) Dehydration Ⅰ: slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 minutes each.

(1.14)复染:用0.5%伊红乙醇液对比染色1min。(1.14) Counterstaining: contrast staining with 0.5% eosin ethanol solution for 1 min.

(1.15)脱水Ⅱ:将切片放入95%乙醇中洗去多余的红色,然后放入无水乙醇中3-5min。最后用吸水纸吸干多余的乙醇。(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)透明:切片放入二甲苯Ⅰ、Ⅱ中各3-5min。(1.16) Transparency: Place the slices in xylene I and II for 3-5 minutes each.

(1.17)封藏:中性树胶封存(1.17) Sealing: neutral gum sealing

(1.18)获取图像:将样本置于显微镜下,通过相机拍摄多个视野的样本显微图像,图像中应加入标尺。(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)肌纤维参数的图像测量(2) Image measurement of muscle fiber parameters

(2.1)将拍摄得到的彩色图像I转化为灰度图像G,然后对G进行阈值分割得到二值图像B1。B1中,深色的肌纤维细胞区域为黑色,背景区域为白色;(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)对B1进行连通域标记,然后去除面积小于给定阈值TA1的那些白色区域。求取区域去除之后的二值图像的反色图像,得到B2(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)对B2进行连通域标记,得到标记图像L1,然后去除L1中的贴边区域,即区域中至少有一个像素点落在了图像的外缘(即图像的最上和最下行以及最左和最右列)上,得到标记图像L2;贴边区域即指个别肌纤维细胞不是全部在图像L1的范围内,而是一部分在图像L1内,另一部分在图像L1外,在图像L1内的那部分不完整的肌纤维细胞区域即所说的贴边区域。(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)去除L2中面积小于给定阈值TA2或大于给定阈值TA3的区域,得到标记图像L3(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)对L3中的每个区域,计算其面积与凸包面积之比r,去除L3中r值小于给定阈值Tacar的区域,得到标记图像L4(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)对L4中的每个区域,对其进行孔洞填充,即得猪肉的肌纤维区域;(2.6) For each area in L 4 , it is filled with holes to obtain the muscle fiber area of pork;

(2.7)计算肌纤维区域的面积(即像素点个数)AI(2.7) calculate the area (i.e. the number of pixels) A I of the muscle fiber region;

(2.8)提取肌纤维区域的轮廓,计算轮廓上每对相异点之间的距离,以所有这些点对的距离的最大值为肌纤维的直径(以像素为单位)dI(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)根据图像中所给的标尺,计算图像中1像素长度所对应的实际长度s,于是肌纤维的实际面积为A=AI·s2,实际直径为d=dI·s。(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.

本发明虽然是针对猪肉的肌纤维参数测量所提,但是如果是其他肉类的肌纤维参数测量,如果这些肉类的肌纤维形态与猪肉的类似,则本发明的方法同样适用,因此应视为本发明所涵盖的范围。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.

本发明所指的肌纤维参数,主要指肌纤维横断面的面积和直径测量。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.

如上所述,对本发明的实施实例进行了详细的说明,但只要实质上没有脱离本发明的特点及效果,是可以有很多变形的,这对本领域的技术人员来说是显而易见的,因此,这些变形的实例也全部包含在本发明的保护范围之内。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)

1.一种肌纤维细胞参数的图像测量方法,其特征在于,包括如下步骤:1. an image measurement method of muscle fiber cell parameter, is characterized in that, comprises the steps: (1)获得肌纤维的图像和图像的标尺;(1) obtain the image of the muscle fiber and the ruler of the image; (2)肌纤维参数的图像测量:(2) Image measurement of muscle fiber parameters: (2.1)将拍摄得到的彩色图像I转化为灰度图像G,然后对G进行阈值分割得到二值图像B1;B1中,深色的肌纤维细胞区域为黑色,背景区域为白色;(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)对B1进行连通域标记,然后去除面积小于给定阈值TA1的白色区域;求取白色区域去除之后的二值图像的反色图像,得到B2(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)对B2进行连通域标记,得到标记图像L1,然后去除L1中的贴边区域,得到标记图像L2(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)去除L2中面积小于给定阈值TA2或大于给定阈值TA3的区域,得到标记图像L3(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)对L3中的每个区域,计算其面积与凸包面积之比r,去除L3中r值小于给定阈值Tacar的区域,得到标记图像L4(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)对L4中的每个区域,进行孔洞填充,即得肌纤维区域;(2.6) Carry out hole filling for each area in L 4 to obtain the muscle fiber area; (2.7)根据肌纤维区域计算得到肌纤维细胞参数。(2.7) Calculate the muscle fiber cell parameters according to the muscle fiber area. 2.如权利要求1所述的肌纤维细胞参数的图像测量方法,其特征在于,步骤(2.7)中,所述肌纤维细胞参数包括肌纤维的实际面积,肌纤维的实际面积计算方法如下: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: 计算肌纤维区域的相对面积即肌纤维区域内的像素点个数AI;根据图像中所给的标尺,计算图像中1像素长度所对应的实际长度s,于是肌纤维的实际面积A=AI·s2Calculate 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.如权利要求1所述的肌纤维细胞参数的图像测量方法,其特征在于,步骤(2.7)中,所述肌纤维细胞参数包括肌纤维的实际直径,肌纤维的实际直径计算方法如下: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: 提取肌纤维区域的轮廓,计算轮廓上每对相异点之间的距离,以所有相异点对的距离的最大值为肌纤维的直径dI,单位为像素;据图像中所给的标尺,计算图像中1像素长度所对应的实际长度s,于是肌纤维的实际直径d=dI·s。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.如权利要求1所述的肌纤维细胞参数的图像测量方法,其特征在于,步骤(2.3)中的贴边区域即若某个肌纤维细胞区域中至少有一个像素点落在了图像的外缘,即图像的最上、最下行、最左列或最右列外的区域,则此肌纤维细胞在图像L1内的区域称作贴边区域。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.如权利要求1所述的肌纤维细胞参数的图像测量方法,其特征在于,所述肌纤维细胞为猪肉纤维细胞。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.如权利要求1所述的肌纤维细胞参数的图像测量方法,其特征在于,步骤(1)中,肌纤维的图像通过相机拍摄多个视野的样本显微图像获得。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.如权利要求6所述的肌纤维细胞参数的图像测量方法,其特征在于,所述样本通过以下步骤制得: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)固定:用4%的多聚甲醛固定样本24小时;(1.1) Fix: fix the sample with 4% paraformaldehyde for 24 hours; (1.2)脱水:将洗好后的样品放入为70%、80%、90%、100%、100%的酒精中分别经过过夜、8小时、8小时、1小时、1小时浸泡以脱去样品中的水分;(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)透明:以1:1的二甲苯:无水酒精浸泡1小时,再用二甲苯浸泡1小时,以置换出组织块中的无水酒精;(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)浸蜡和包埋:将透明好的样品放入1:1的二甲苯:石蜡中过渡30分钟,再将其放入装有石蜡的蜡杯中30分钟,需在温箱中将石蜡熔化后进行,温箱温度不得高于56℃,将浸好蜡的组织块放入盛有熔化石蜡的纸盒内,使组织块包埋于石蜡中,迅速放在水盆中,待其冷却后备用;(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)切片:将制好的组织蜡块经修切后粘附于台木后用切片机切成6μm厚的薄片,薄片连续成蜡带状;(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)展片和粘片:将切成的蜡带展于30℃的温水中,再以1:1的鸡蛋清:甘油混合剂为粘附剂,将组织薄片粘附于载玻片上,再将其置于37℃温箱中烘干,不少于24h;(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)脱蜡复水:将水浴锅温度调至60℃,待水温控制在60℃时,将切片连同切片架放入一个干燥的染色缸内,放入水浴锅中,盖上盖子,30min至蜡熔化;(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)石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各5min,然后放入100%、95%、90%、80%、70%各级浓度的酒精溶液中各3-5min,再放入蒸馏水中3min;(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)染色:切片放入苏木精中染色15min;(1.9) Staining: Put the slices into hematoxylin and stain for 15 minutes; (1.10)水洗:用自来水流水冲洗15min;使切片颜色变蓝;(1.10) Washing: Rinse with running water for 15 minutes; make the slices turn blue; (1.11)分化:将切片放入1%盐酸乙醇液中褪色,2秒至数十秒钟;见切片变红,颜色较浅即可;(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)漂洗:切片再放入自来水流水中使其恢复蓝色;(1.12) Rinsing: put the slices into running water to make them turn blue again; (1.13)脱水Ⅰ:切片入50%乙醇→70%乙醇→80%乙醇中各3-5min;(1.13) Dehydration Ⅰ: slice into 50% ethanol → 70% ethanol → 80% ethanol for 3-5 minutes each; (1.14)复染:用0.5%伊红乙醇液对比染色1min;(1.14) Counterstaining: contrast staining with 0.5% eosin ethanol solution for 1 min; (1.15)脱水Ⅱ:将切片放入95%乙醇中洗去多余的红色,然后放入无水乙醇中3-5min;最后用吸水纸吸干多余的乙醇;(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)透明:切片放入二甲苯Ⅰ、Ⅱ中各3-5min;(1.16) Transparency: Put the slices into xylene I and II for 3-5 minutes each; (1.17)封藏:中性树胶封存。(1.17) Sealing: neutral gum sealing.
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