WO2017092136A1 - 一种基于光干涉技术的叶片表面微结构快速测量方法 - Google Patents

一种基于光干涉技术的叶片表面微结构快速测量方法 Download PDF

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WO2017092136A1
WO2017092136A1 PCT/CN2015/099648 CN2015099648W WO2017092136A1 WO 2017092136 A1 WO2017092136 A1 WO 2017092136A1 CN 2015099648 W CN2015099648 W CN 2015099648W WO 2017092136 A1 WO2017092136 A1 WO 2017092136A1
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sample
blade
measurement
leaf
microstructure
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李青林
毛罕平
左志宇
倪纪恒
孙俊
张晓东
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Jiangsu University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N2021/8466—Investigation of vegetal material, e.g. leaves, plants, fruits

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  • the invention relates to the field of plant physiology, in particular to a rapid measurement technology for the surface microstructure of a blade.
  • the existing surface microstructure detection methods of the object mainly include: scanning electron microscope (SEM) imaging method, atomic force microscopic imaging method and optical interference imaging method.
  • the research method for the surface microstructure of the blade is mainly scanning electron microscopy (SEM) imaging.
  • SEM scanning electron microscopy
  • the secondary electrons with morphological and structural information on the sample are clicked point by point.
  • the surface is bombarded and the high resolution image of the sample surface is displayed on the screen after being processed by the detector. Because plant leaves contain more water and have poor conductivity, they need to be dried, sprayed, etc. before imaging, and the pretreatment is complicated and the treatment cycle is long.
  • the core structure of atomic force microscopy is a very force-sensitive microcantilever with a tiny probe at its tip.
  • the detector converts the deformation signal of the microcantilever into a photoelectric signal for amplification, and obtains a weakly varying signal of the force between the atoms, thereby obtaining distribution information of the force, thereby obtaining the force distribution information at a nanometer resolution, thereby being nanoscale.
  • the resolution obtains the surface topography. The leaves of most plants are distributed with fluff.
  • the height and straightness of the fluff are generally from a few micrometers to several tens of micrometers. Some of the surface of the leaves can reach a few hundred micrometers in height, and the size is much larger than that of the scanning probe. Scanning will cause problems such as breakage of the probe, so it is not suitable to measure the surface structure of the blade by this method.
  • Optical interferometry is a method for measuring the optical path difference using the principle of optical interference to determine the surface microstructure of the object under test. law. Any change in the optical path difference between the two coherent beams can very sensitively cause the movement of the interference fringes, and the optical path change of a certain beam is caused by the change of the geometric path through which it passes, so the movement of the interference fringes can be measured. The length of the tiny variable.
  • the method has non-contact measurement, fast and high precision, and is widely used in surface microstructure measurement. Currently, the method is mainly used for measurement of material surface and machined surface. However, no research reports on the measurement of the surface microstructure of the blade by this method have been reported.
  • the existing blade surface microstructure measurement method has the problems of complicated pre-processing and long processing period.
  • the method uses optical interference technology to image the surface of the blade and characterize the three-dimensional shape without any pre-processing, which solves the problem that the current measurement method has complicated pre-processing and long processing period.
  • the invention aims to provide a rapid measurement method for the surface microstructure of the blade based on the optical interference technique, so as to realize the rapid and simple acquisition of micro-structure features such as pores and fluff on the surface of the blade; and characterize the microstructure characteristics.
  • the specific technical solutions adopted by the present invention are as follows:
  • a method for rapidly measuring the surface microstructure of plant leaves based on optical interference technology comprising the following steps:
  • Step one sampling and fixing: avoiding the leaf vein sample obtained by cutting the leaf tissue, the leaf sample size is 2cm*2cm, and fixing the leaf sample with a concentration of 4% glutaraldehyde fixing solution; the fixing is The leaves are placed in the solution to maintain their organelle morphology;
  • Step two placing the leaf sample horizontally on the glass slide
  • Step 3 placing the slide glass carrying the blade sample on the stage, so that the bottom surface of the slide surface is in close contact with the upper surface of the stage; the leaf sample is located directly below the lens;
  • Step 4 illuminating the blade sample with an LED light source
  • Step 5 using an optical imaging system to image the surface of the leaf sample of the test area, and determine the scanning distance when the different structures are observed according to different microstructure characteristics of the leaf sample;
  • Step 6 Determine the measurement reference of the surface microstructure of the blade sample, and obtain the main parameters of the measurement system measurement to evaluate the microstructure of the cucumber leaf sample surface, including geometric shape parameters, height parameters, profile parameters, and volume parameters.
  • the second step is specifically: the blade sample face down, so that the blade sample is closely attached to the 25.4 mm ⁇ 76.2 mm slide and is at a level.
  • the step 4 is specifically: selecting high-brightness LED illumination in a given four illumination modes; the four illumination modes are: reflective bright field LED illumination, reflected dark field LED illumination, high-brightness LED illumination, and transmission LED lighting.
  • the step 5 is specifically: when the pores, mesophyll cells and vascular bundles are observed, the height range collected is 20 um; when the fluff is observed, the height range collected is 200 um.
  • the measurement reference is the lowest point of the mesophyll tissue epidermal cells of the leaf sample; and the microstructural features of the surface of the leaf sample are measured by an optical measurement system, including: morphological characteristics of the leaf surface stomata, mesophyll epidermis cells, vascular bundles, and villi;
  • the pores were measured by geometric measurement methods, and the morphology of the pores were measured and characterized by height measurement, profile measurement and volume measurement.
  • the morphology of mesophyll cells was characterized by the volume measurement of mesophyll cells in the specified 50um*50um region to characterize the morphology of mesophyll cells.
  • geometric measurements, profile measurements, and volumetric measurements are used to characterize vascular bundles.
  • the present invention has a beneficial effect.
  • the invention can quickly and easily acquire the surface microstructure of the blade by means of the optical interference technique; the invention explores the expression parameters of the microstructure characteristics of the blade surface from the microscopic scale, and can quantitatively describe the microstructure characteristics of the blade surface; The basis for the evaluation of the growth state of the crop is provided by quantitative analysis of the surface microstructure parameters; the present invention provides a rapid measurement method for the surface microstructure of the blade.
  • FIG. 1 is a schematic diagram of a measuring optical path of a method for measuring the surface microstructure of a blade based on optical interference technology.
  • Figure 2 is a schematic illustration of the measurement principle of the present invention.
  • Figure 3-a is a two-dimensional structure diagram of the surface of the cucumber leaf of the present invention
  • Figure 3-b is a three-dimensional structure diagram of the surface fluff of cucumber leaves of the present invention
  • Figure 3-c is a cloud diagram of the surface velvet height measurement of the cucumber leaf of the present invention.
  • Figure 4-a is a two-dimensional structure diagram of the stomata and mesophyll cells on the surface of cucumber leaves of the present invention.
  • Figure 4-b is a three-dimensional structure diagram of the stomata and mesophyll cells on the surface of cucumber leaves of the present invention.
  • Figure 4-c is a cloud diagram of the stomata and mesophyll cell height measurement on the surface of cucumber leaves of the present invention.
  • the microscope system used in this embodiment is a DSX500 model manufactured by Olympus Corporation of Japan.
  • the measurement system mainly includes an LED light source, and the surface structure of the greenhouse tomato leaves is collected by the microscopic image acquisition system in the microscopic system.
  • the invention was carried out in the glass greenhouse of the Key Laboratory of Modern Agricultural Equipment and Technology of Ministry of Education of Jiangsu University from March 2015 to August 2015, and the cucumber variety was selected from Jinchun No.4.
  • the greenhouse is sampled and immediately put into the pre-formed glutaraldehyde fixing solution.
  • a slide method is adopted, and the sample of the cucumber leaf is placed on the slide and placed on the stage. . That is, it was taken out from the fixing solution and immediately placed on a 25.4 x 76.2 mm glass slide with the blades facing down and the back side facing up, and the blades were placed against the slides to ensure that the mesophyll samples were horizontal.
  • the height range of the collection is 20um, and the acquisition is performed in the fine collection mode.
  • the focus imaging is performed again. At this time, the height range of the collection is set to 200um, and the collection is performed in the fine collection mode. .
  • the observation of the microstructure of the blade surface shows that the optical interference technique can quickly acquire 2D and 3D images of the pores, mesophyll cells, vascular bundles and fluff of the leaf surface, and the image can clearly reflect the various microstructures on the surface of the blade.
  • Morphological parameters of structures such as stomata, mesophyll cells, vascular bundles, and villi are measured, including geometric measurements, height measurements, profile measurements, and volume measurements. At least 10 fields of view were observed for each sample and averaged.
  • Morphological parameters of the pores Because the size and shape of the pores directly affect the physiological state of the crop, geometric measurements are used to measure the size of the pores; height measurements, profile measurements and volumetric measurements are used to characterize the morphology of the pores.
  • Morphological parameters of mesophyll cells Mesophyll epidermal cells are the most abundant cells on the surface of leaves, and the morphology of each cell is different. Therefore, in the evaluation of mesophyll cells, the volume of mesophyll cells in the designated 50um*50um region is used to characterize the mesophyll epidermis. Morphological characteristics of the cells.
  • Morphological parameters of 3-dimensional tube bundles The distribution of vascular bundles on the surface of the blade has certain regularity, but the diameter of the vascular bundle varies greatly depending on its distribution position. Therefore, geometric measurement, profile measurement and volume measurement are selected to characterize the vascular bundle. Morphological parameters.
  • the height of the fluff is much greater than the height of other microstructures on the surface of the blade, so the height and volume parameters are used to characterize its morphology.

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Abstract

一种基于光干涉技术的叶片表面微结构快速测量方法，采用光学测量方法代替扫描电镜技术获取叶片表面的微观形态特征，并加以定量表征，解决了叶片表面特征无法直接快速获取、定量进行表征的问题，具有快速、简单易行、可以定量分析等优点，可应用于叶片表面微结构快速测量。

Description

一种基于光干涉技术的叶片表面微结构快速测量方法 技术领域
本发明涉及一种植物生理领域，具体涉及一种叶片表面微结构快速测量技术。
背景技术
植物营养学的研究已经证明，叶片作为植物的营养器官，其生长发育状态直接受养分供给的影响。营养水平变化影响植物正常代谢活动，引起植物叶片表面气孔、绒毛、维管束等微结构形态和密度的变化。因此，获取叶片表面的微结构特征对于作物养分诊断和科学研究具有重要的意义。
叶片表面微结构内活性物质、霉、蛋白质等的变化，造成叶片组织细胞形态、细胞排列以及细胞内含物等一系列微观结构信息变化，当这些微观变化积累到一定程度才会最终引起叶片颜色、大小等宏观特征的变化。因此，可以通过观测叶片表面微观结构特征变化，对作物的养分水平进行早期诊断。目前常用的方法是扫描电镜方法获取叶片表面微结构，该方法前处理复杂，处理周期长，不能实时了解作物的养分情况。本发明试图提供一种叶片表面微结构快速测量方法。
现有物体表面微结构检测方法主要有：扫描电子显微镜(SEM)成像法，原子力显微成像法以及光干涉成像法等。
对于叶片表面微结构的研究方法目前主要是扫描电镜(SEM)成像法，扫描电子显微镜是将电子束在样品上进行动态扫描时，将样品上带有形态和结构信息的二次电子逐点逐行地轰击出表面，经检测器处理后再屏幕上显示样品表面的高分辨形貌图像。因为植物叶片含水分较多，且导电性差，因此在成像之前需要进行干燥、喷金等处理，前处理复杂，处理周期长。
原子力显微镜(atomic force microscopy，AFM)的核心结构时一个对力非常敏感的微悬臂，其尖端有一个微小的探针。当探针靠近样品表面时，探针尖端的原子与样品表面的原子之间产生极其微弱的作用力，从而使微悬臂弯曲。探测器将微悬臂的形变信号转换成光电信号进行放大，就可以得到原子之间力的微弱变化信号，获得作用力的分布信息，从而以纳米级分辨率获得作用力分布信息，从而以纳米级分辨率获得表面形貌结构。大多数植物叶片表面都分布有绒毛，绒毛的高度和直经一般从几微米到几十微米，有的叶片表面绒毛高度可以达到几百微米，尺寸远远大于扫描探针，若对叶片表面进行扫描，会造成探针折断等问题，因此不适合用该方法进行叶片表面结构的测量。
光干涉测量，是一种利用光干涉原理测量光程差从而测定被测对象表面微结构的方 法。两束相干光间光程差的任何变化会非常灵敏地导致干涉条纹的移动，而某一束光的光程变化是由它通过的几何路程的变化引起，所以干涉条纹的移动变化可测量几何长度的微小该变量。该方法具有非接触测量、快速、高精度，在表面微结构测量方面应用广泛，目前该方法主要用于材料表面和机械加工表面的测量中。但未见借助该方法测量叶片表面微结构的研究报道。
现有的叶片表面微结构测量方法存在前处理复杂、处理周期长的问题。本方法采用光干涉技术对叶片表面进行成像和三维形态的表征，无需任何前处理，解决了目前测量方法前处理复杂，处理周期长的问题。
发明内容
本发明目的在于提供一种基于光干涉技术的叶片表面微结构快速测量方法，以实现快速、简单地采集叶片表面气孔、绒毛等微结构特征；并对微结构特征进行表征。为了解决以上技术问题，本发明采用的具体技术方案如下：
一种基于光干涉技术的植物叶片表面微结构快速测量方法，其特征在于包括以下步骤：
步骤一，取样并固定：避开主脉切取叶片组织得叶片样本，叶片样本大小为2cm*2cm，并对叶片样本用浓度为4％的戊二醛固定液进行固定；所述的固定为将叶片放到溶液里面，保持其细胞器形态；
步骤二，将所述叶片样本水平置于载玻片上；
步骤三：将载有叶片样本的载玻片放置于载物台上，使载玻片底面紧贴载物台上表面；使叶片样本位于镜头正下方；
步骤四：采用LED光源对所述叶片样本进行照明；
步骤五：利用光学成像系统，对待测区的叶片样本表面进行成像，根据叶片样本的不同微结构特征，确定不同结构观察时的扫描距离；
步骤六：确定叶片样本表面微结构的测量基准，获取测量系统测量能评价黄瓜叶片样本表面各微结构的主要参数，包括几何形态参数、高度参数、剖面参数、体积参数。
所述步骤二具体为：将叶片样本正面朝下，使叶片样本紧贴于25.4mm×76.2mm载玻片，并处于水平。
所述步骤四具体为：在给定的四种照明方式中选择高亮度LED照明；所述四种照明方式为：反射明视场LED照明、反射暗视场LED照明、高亮度LED照明和透射LED照明。
所述步骤五具体为：观察气孔、叶肉细胞以及维管束时，采集的高度范围是20um；需要观察绒毛时，采集的高度范围是200um。
所述测量基准为叶片样本的叶肉组织表皮细胞的最低点；利用光学测量系统，测量所述叶片样本表面的微结构特征包括：叶片表面气孔、叶肉表皮细胞、维管束、绒毛的形态特征；所述气孔用几何测量方法来测量其大小，用高度测量、剖面测量和体积测量方法来测量气孔形态并加以表征；叶肉细胞形态用指定50um*50um区域内叶肉细胞的体积测量来表征叶肉细胞的形态特征；用几何测量、剖面测量和体积测量的方法来表征维管束的特征。
本发明具有有益效果。本发明借助光干涉技术，可以快速、简便地获取叶片表面微结构；本发明从微观尺度出发，探求了叶片表面微结构特征的表达参数，能定量描述叶片表面的各微结构特征；本发明可以通过对表面微结构参数的定量分析，为作物的生长状态评价提供依据；本发明提供了一种叶片表面微结构的快速测量方法。
附图说明
图1本发明一种基于光干涉技术的叶片表面微结构测量方法的测量光路示意图。
图2是本发明测量原理示意图。
图3-a是本发明黄瓜叶片表面绒毛二维结构图
图3-b是本发明黄瓜叶片表面绒毛三维结构图
图3-c是本发明黄瓜叶片表面绒高度测量云图
图4-a是本发明黄瓜叶片表面气孔和叶肉细胞二维结构图
图4-b是本发明黄瓜叶片表面气孔和叶肉细胞三维结构图
图4-c是本发明黄瓜叶片表面气孔和叶肉细胞高度测量云图
具体实施方式
下面以黄瓜为例，结合附图和具体实施例对本发明的技术方案做进一步详细说明。
本实施例中所采用的显微镜系统是由日本奥利巴斯公司生产的DSX500型，该测量系统主要包括LED光源，利用该显微系统中的显微图像采集系统采集温室番茄叶片的表面结构。本发明于2015年3月至2015年8月在江苏大学现代农业装备与技术教育部重点实验室玻璃温室中进行实验，黄瓜品种选用津春四号。
(1)样本表面微结构固定
为保证黄瓜叶片在取样后观察时保持生长时的表面微结构状态，在温室取样后立刻投入事先配好的戊二醛固定液中。
(2)放置
因为植物组织较柔软的特殊性，不能保证和金属样本一样紧贴载物台表面，因此本发明中采用了载玻片的方法，将黄瓜叶片样本放在载玻片之后，置于载物台。也即从固定液中取出，立刻置于25.4×76.2mm的载玻片上，叶片正面朝下，背面朝上，并使叶片紧贴载玻片以保证叶肉样本处于水平状态。
(3)调整
调整物镜和载物台之间的距离(Z向)使图像聚焦，根据测量对象的不同，选择合适的采集范围。因为黄瓜叶片表面绒毛高度远远大于气孔、叶肉细胞以及维管束的高度，所以在观察时，需要调整镜头Z方向距离。观察气孔、叶肉细胞以及维管束时，采集的高度范围是20um，用精细采集模式进行采集；需要观察绒毛时，重新进行聚焦成像，此时设置采集的高度范围是200um，用精细采集模式进行采集。
(4)叶片表面结构观察
在显微系统下观察叶片表面气孔、叶肉细胞、维管束和绒毛的几何特征，分别进行2D和3D成像，选取典型视野拍照(图3和图4)。
叶片表面显微结构观察表明，光干涉技术能快速获取叶片表面气孔、叶肉细胞、维管束以及绒毛等结构的2D和3D图像，图像能清晰地反映叶片表面的各种微结构。
(5)测量叶片各特征结构的形态参数
测量气孔、叶肉细胞、维管束以及绒毛等结构的形态参数，包括几何测量、高度测量、剖面测量、体积测量。每个样本观察至少统计10个视野，取平均值。
①气孔的形态参数：因为气孔的大小和形态直接影响作物的生理状态，所以用几何测量方法来测量气孔的大小；用高度测量、剖面测量和体积测量方法来综合表征气孔的形态特征。
②叶肉细胞的形态参数：叶肉表皮细胞是叶片表面分布最丰富的细胞，各个细胞的形态存在一定差异，因此在评价叶肉细胞是，采用指定50um*50um区域叶肉表皮细胞的体积测量在表征叶肉表皮细胞的形态特征。
③维管束的形态参数：叶片表面维管束分布具有一定的规律性，但维管束直径因其分布位置的不同，存在较大差异，所以选择几何测量、剖面测量和体积测量的方法来表征维管束的形态参数。
④绒毛的形态参数：绒毛的高度远远大于叶片表面的其它微结构的高度，因此用高度和体积参数来表征其形态。

Claims (5)

  1. 一种基于光干涉技术的植物叶片表面微结构快速测量方法，其特征在于包括以下步骤：
    步骤一，取样并固定：避开主脉切取叶片组织得叶片样本，叶片样本大小为2cm*2cm，并对叶片样本用浓度为4％的戊二醛固定液进行固定；所述的固定为将叶片放到溶液里面，保持其细胞器形态；
    步骤二，将所述叶片样本水平置于载玻片上；
    步骤三：将载有叶片样本的载玻片放置于载物台上，使载玻片底面紧贴载物台上表面；使叶片样本位于镜头正下方；
    步骤四：采用LED光源对所述叶片样本进行照明；
    步骤五：利用光学成像系统，对待测区的叶片样本表面进行成像，根据叶片样本的不同微结构特征，确定不同结构观察时的扫描距离；
    步骤六：确定叶片样本表面微结构的测量基准，获取测量系统测量能评价黄瓜叶片样本表面各微结构的主要参数，包括几何形态参数、高度参数、剖面参数、体积参数。
  2. 根据权利要求1所述的一种基于光干涉技术的叶片表面微结构测量方法，其特征在于所述步骤二具体为：将叶片样本正面朝下，使叶片样本紧贴于25.4mm×76.2mm载玻片，并处于水平。
  3. 根据权利要求1所述的一种基于光干涉技术的叶片表面微结构测量方法，其特征在于所述步骤四具体为：在给定的四种照明方式中选择高亮度LED照明；所述四种照明方式为：反射明视场LED照明、反射暗视场LED照明、高亮度LED照明和透射LED照明。
  4. 根据权利要求1所述的一种基于光干涉技术的叶片表面微结构测量方法，其特征在于所述步骤五具体为：观察气孔、叶肉细胞以及维管束时，采集的高度范围是20um；需要观察绒毛时，采集的高度范围是200um。
  5. 根据权利要求1所述的一种基于光干涉技术的叶片表面微结构测量方法，其特征在于：所述测量基准为叶片样本的叶肉组织表皮细胞的最低点；利用光学测量系统，测量所述叶片样本表面的微结构特征包括：叶片表面气孔、叶肉表皮细胞、维管束、绒毛的形态特征；所述气孔用几何测量方法来测量其大小，用高度测量、剖面测量和体积测量方法来测量气孔形态并加以表征；叶肉细胞形态用指定50um*50um区域内叶肉细胞的体积测量来表征叶肉细胞的形态特征；用几何测量、剖面测量和体积测量的方法来表征维管束的特征。
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