A method for preparing a pollen scanning electron microscope sample

Abstract

The application discloses a pollen scanning electron microscope sample preparation method, which comprises the following steps: putting pollen into a glass dish, placing the pollen at a position 15-25 cm below the center of an infrared baking lamp, drying the pollen for 100-150 min, sputtering an alloy on the surface of the dried pollen sample, and observing the microstructure of the pollen sample by using a scanning electron microscope. The method established by the application introduces an infrared baking lamp, replaces a mechanically complicated supercritical drying instrument for drying the sample, omits the dehydration and drying steps, and saves the fixing and dehydration time. Meanwhile, the method has little influence on the sample appearance change and cannot cause the loss of the pollen sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sample preparation for scanning electron microscope microscopic observation, and particularly relates to a pollen scanning electron microscope sample preparation method. BACKGROUND

[0002] Pollen is one of the carriers of plant genetic information, and is the basis of plant growth, inheritance and variation. Different types of plant pollen will form unique and stable morphological characteristics, and the inherent outline, ornamentation and number of germination pores reflect the unique biological information of the species. The morphologies of pollen of the same group of plants are similar. Classification and identification using these inherent characteristics have important scientific value and can lay a theoretical foundation for future research on plant phylogeny, classification and genetic relationship. Traditional pollen identification and quantitative analysis is very time-consuming and has low resolution in identifying species. Under an optical microscope, pollen dispersed in sediments can only be identified at the genus level, and some can only be identified at the family level. Scanning electron microscopy is widely used in the observation of plant spore micro-morphology due to its high resolution and stereoscopic and intuitive image, and is an important tool for studying pollen morphology.

[0003] In a scanning electron microscope, the quality of sample preparation directly affects the observation effect and correct interpretation of the electron microscopic image. Therefore, pollen samples need to be dehydrated and dried before being attached to the stage and sprayed with gold. Drying is a critical step in sample preparation, and improper drying can directly affect the clarity and accuracy of the image and cause contamination of the objective lens, electron gun and other components in the electron microscope. Through literature review, common scanning electron microscope sample preparation methods include conventional biological electron microscope preparation method, direct drying preparation method, conductive double-sided tape breaking method and fresh material direct gold spraying.

[0004] The conventional biological electron microscope preparation method, also known as biological fixation method, uses chemical reagents to fix the sample morphology, and through gradient dehydration with ethanol, drying and other preparation processes, the sample is prepared for scanning electron microscope observation. This method is most commonly used in electron microscope preparation, and the effect of the obtained image is good, but the steps are complicated and time-consuming. The direct drying preparation method, also known as natural drying method, can observe the sample after being placed in a clean environment and naturally dried. This method is simple to operate and saves the fixation and dehydration steps, but the sample is prone to deformation, and is commonly used for samples that are not prone to deformation. The conductive double-sided tape breaking method involves attaching the sample to the conductive tape, gently clamping the sample with tweezers, pulling it upwards, and sticking the broken part of the sample to the conductive tape. This method can observe the interior of the pollen, but it has strict technical requirements for the operator and is difficult to prepare samples. The fresh material direct gold spraying method is simple to operate and saves time, but it may affect the morphology of the sample during sample preparation, and requires high sample shape setting.

[0005] The pretreatment of the sample for scanning electron microscopy is very important, and in addition to biological fixation, the selection of drying method also has certain influence on the morphology of the sample and the drying result. Commonly used drying methods include CO2 critical point drying method, freeze drying method and direct drying method. The CO2 critical point drying method has strict requirements and complicated steps, and improper operation of a small part may cause mechanical damage to the sample and result in drying failure; the freeze drying method uses low temperature to harden the sample, and directly sublimes water under high vacuum condition, thereby avoiding damage of surface tension to the sample, but the sample may be damaged by freezing. The direct drying method, also known as natural drying method, can be observed after the sample is naturally dried, and the operation is simple, but the sample is easy to deform and the drying time is long. SUMMARY

[0006] In view of the above problems in the prior art, the purpose of the present application is to provide a pollen scanning electron microscopy sample preparation method to solve the problems of long drying time, sample deformation and loss in the preparation process of the existing pollen scanning electron microscopy sample.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application provides a pollen scanning electron microscopy sample preparation method, which comprises the following steps:

[0009] The pollen is placed in a glass dish, and is dried for 100-150 min under the center of an infrared baking lamp at a distance of 15-25 cm. An alloy is sputtered on the surface of the dried pollen sample, and the microstructure is observed by scanning electron microscopy.

[0010] Further, the method specifically comprises the following steps:

[0011] Step 1. The anther is gently taken out with tweezers, and the pollen is retained on the anther to avoid the pollen from scattering and facilitate the adhesion of the sample to the table. The anther is placed in a glass dish, and is labeled.

[0012] Step 2. The glass dish containing the pollen is placed under the center of a near-infrared baking lamp at a distance of 20 cm, and is dried for 90 min. Then the glass dish is moved to the edge, and the sample is dried for 30 min by using the edge temperature to assist the sample in drying by radiation and heat transfer.

[0013] Step 3. A clean weighing paper is taken, and after the sample is dried to have no extra moisture on the outside, the anther is taken out with tweezers, and is gently tapped on the wrist. The pollen is evenly dropped on the light surface of the weighing paper by slight vibration, and then the sample table with conductive glue is inverted and the pollen is evenly adhered.

[0014] Step 4. The pollen sample table is placed in an ion sputtering instrument one by one, and an alloy is sputtered on the surface of the sample for 60 s.

[0015] Step 5, the pollen sample is observed by scanning electron microscopy to observe its micro-morphology, and a uniform acceleration voltage of 5kv is set.

[0016] Further, the infrared spectrum ranges from 0.7 μm to 2.5 μm.

[0017] The present application has the following advantages:

[0018] The method established by the present application introduces an infrared baking lamp, replaces the mechanical supercritical drying instrument for drying the sample, omits the dehydration and drying steps, and saves the fixing and dehydration time. At the same time, the sample morphology change is less affected, and the pollen sample is not lost. BRIEF DESCRIPTION OF DRAWINGS

[0019] Fig. 1 is a sample morphology diagram of pollen under far infrared radiation;

[0020] Fig. 2 is a sample morphology diagram of pollen under mid-infrared radiation;

[0021] Fig. 3 is a sample morphology diagram of pollen under near-infrared radiation;

[0022] Fig. 4 is a sample morphology diagram of 'Taigong No. 1' pollen under near-infrared radiation;

[0023] Fig. 5 is a sample morphology diagram of 'Jinhuang mango' pollen under near-infrared radiation;

[0024] Fig. 6 is a sample morphology diagram of 'Repin No. 16' pollen under near-infrared radiation. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application are described below to facilitate those skilled in the art to understand the present application, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, as long as various changes are within the spirit and scope of the present application defined and determined by the appended claims, these changes are obvious, and all applications utilizing the concept of the present application are within the scope of protection.

[0026] EMBODIMENT

[0027] A pollen scanning electron microscope sample preparation method, the method comprising the following steps:

[0028] Step 1, use tweezers to gently take anther, keep pollen on the anther of stamen to avoid pollen from scattering, facilitate sticking to the table; put it in a glass dish and mark it.

[0029] Step 2, place the glass dish containing the pollen under the center of the near-infrared baking lamp at a distance of 20 cm and dry for about 90 min, at this time the center of the infrared baking lamp can reach 45 DEG C, the penetration of short-wave infrared is stronger, so that the inside of the pollen is thoroughly heated. Then move the glass dish to the edge, use the edge temperature (about 30-40 DEG C) to assist the pollen to dry by radiation and its own heat transfer to dry the sample, about 30 min. Preferably, the infrared spectrum range: 0.7-2.5 um.

[0030] Step 3, take a clean weighing paper, after the sample is dried to the outside without excess moisture, use the forceps to take out the anther, pat the wrist, evenly fall the pollen on the weighing paper by slight vibration, then turn over the sample stage with conductive glue and evenly stick the pollen;

[0031] Step 4, place the pollen sample stage into the ion sputtering instrument one by one, sputter the alloy on the surface of the sample for 60 s;

[0032] Step 5, observe the micro-morphology of the pollen sample by scanning electron microscope, set the uniform acceleration voltage to 5 kv.

[0033] The application establishes a convenient and fast method for preparing pollen scanning electron microscope samples, and is applied to scanning electron microscope observation of mango pollen. In the embodiment, the male pollen of mango germplasm is used as the material, the dried pollen sample is prepared by using infrared radiation, and the wavelength range, time range and distance range of drying are optimized. The results show that the sample is placed at a distance of 20 cm from the infrared generating point, the near-infrared wavelength range (2.5-0.7 um) is used, and the whole imaging effect is best after drying the sample for 120 min. The method can not only more accurately display the morphological characteristics of the pollen, but also can reduce the loss of the pollen sample and save the sample preparation time. At the same time, different varieties of mango pollen are prepared by using the method and morphological observation is carried out, and good imaging effect is obtained.

[0034] Principle: Energy is mainly in the form of radiation directly acting on the material. In infrared radiation, due to the penetration of infrared rays, the energy is first accumulated in the interior of the material. When the atoms and molecules in the plant absorb the energy of the infrared rays, the movement of the particles is intensified, the energy level of the molecules is changed, so that the interior of the material is heated. Due to the continuous evaporation of water, heat is absorbed, the external temperature is lowered, and an internal high-external low temperature gradient is formed. According to the second law of thermodynamics, heat can spontaneously transfer from a high-temperature object to a low-temperature object. At this time, the heat in the material is the conduction medium, and the heat transfer is from the inside to the outside along the temperature gradient, thereby realizing the heating of the whole material. Therefore, under the joint action of the internal high-external low temperature gradient and the humidity gradient, the infrared radiation drying can greatly improve the drying rate of the material.

[0035] The best existing technology is the conventional biological sample preparation method, which uses chemical reagents to fix the morphology of the sample, and then gradually performs gradient dehydration and drying steps. The method established in the present application introduces an infrared baking lamp, which replaces the mechanical supercritical dryer for drying the sample, eliminates the dehydration and drying steps, and saves the fixing and dehydration time. At the same time, the sample morphology change is less affected, and the pollen sample loss is not caused.

[0036] Selection of infrared wavelength: Infrared is an electromagnetic wave between visible light and microwave, with a wavelength range of 0.7-1000 μm. It is divided into near-infrared, mid-infrared and far-infrared; the wavelength ranges are (2.5 μm ~ 0.7 μm), (25 μm ~ 2.5 μm), (500 μm ~ 25 μm), respectively. Therefore, the above three wavelength ranges are selected for drying the sample, and the maximum, minimum and intermediate wavelengths are selected for each range. When the water content reaches below 15%, stop drying, and compare the drying time and sample surface morphology characteristics.

[0037] Establishment of early instrument conditions: The samples were placed at a distance of 10 cm and 20 cm from the infrared generating point, and the drying time was set to 60 min, 120 min and 180 min. During the experiment, a thermometer was set to detect the surface temperature of the object during drying, and the drying degree and sample surface morphology change were observed.

[0038] Reference Figs. 1-3 The results show that the energy transmitted by near-infrared is appropriate, the sample morphology is complete, and the drying time is the shortest; the sample surface is slightly shrunk by mid-infrared drying, without excessive damage, but the drying time is longer than that of infrared; far-infrared causes the sample surface to shrink, and there is a situation of partial sample collapse. Since the pollen sample has a small volume, far-infrared has a strong thermal effect and is not suitable for drying pollen samples. Therefore, the near-infrared wavelength range is selected for drying the sample.

[0039] The sample at a distance of 10 cm from the infrared generating point heats up too quickly, reaching 50℃ within 10 min, and the radiation intensity is large, which can easily cause the sample to deform due to over-drying; while the sample at a distance of 20 cm heats up slowly, reaching the appropriate drying temperature of pollen (45℃) at 60-90 min. There is little difference between the samples dried for 120 min and 180 min observed by scanning electron microscopy, and the 180 min time-consuming is long, so 120 min is selected as the optimal drying time.

[0040] Reference Figs. 4-6 The method is applied to the preparation of scanning electron microscopy samples of "Taikang No. 1", "Jinhuang Mango", and "Repin 16" mango pollen, and the results show that the sample itself can maintain its morphological characteristics without drying, collapsing, etc.

[0041] Through comparison of different pretreatment methods, freeze-drying method is easy to cause the rupture of pollen germination opening, supercritical drying method preserves the sample itself morphology, but the viscosity is strong, and the single pollen sample morphology is not complete. The infrared oven lamp method avoids the rupture of the sample germination opening, saves the tedious fixation and dehydration steps, and has no negative effect on the sample morphology.

[0042] The infrared drying method of the present application adds stable temperature and radiation intensity on the basis of natural air drying, which can quickly dry the sample and achieve "what is made is what is obtained", avoids sample loss, and ensures the sample morphology is not affected. Similar solutions use an oven to dry, but tests show that the oven-dried samples are prone to cracks. So far, no one has used infrared radiation in an infrared oven lamp to dry scanning electron microscope samples. This method is simple to operate and suitable for small-area liquid samples, which can not only avoid sample loss but also shorten the sample preparation time and reduce experimental costs (other drying methods require more special gases and chemical reagents). This method can be widely used in electron microscope sample preparation methods.

[0043] The Chinese invention patent with application number 201911219775.7 discloses a method for low-temperature vacuum suspension pyrolysis of pollen, pyrolysis of pollen and products, wherein the pollen is dried by infrared rays, which has good controllability, rapid heating and short drying time. The temperature of the infrared drying is 25-30℃, and the time is 3-5min, which can reduce the water content of the original pollen from 20%-30% to ≤5%. The effective nutritional ingredients and inherent color of the pollen can be maintained during drying, and mold growth and deterioration can be prevented.

[0044] The Chinese invention patent with application number 201911219775.7 uses infrared drying of pollen, which aims to reduce the water content of pollen and break the pollen wall, but does not optimize the drying conditions.

[0045] The application is scanning electron microscope research on spore morphology, by using infrared drying pollen, not only can reduce the water content of pollen, but also can protect the complete morphological characteristics of pollen. And, according to the sample requirements of scanning electron microscope, the sample preparation process of pollen sample is optimized. By comparing different wavelengths (2.5-0.7, 25-2.5, 500-25), the distance between sample and energy point (10 cm, 20 cm) and the drying time (60 min, 120 min, 180 min), the best sample preparation condition is determined. In the application, the glass dish is moved to the edge at the last node of drying, the purpose is to use the heat transfer of the radiation drying itself to assist the drying of pollen, to avoid the surface temperature too high and cause damage to the surface morphology of pollen. The application provides a preparation method of pollen scanning electron microscope sample, which can be applied to the surface micro-morphology research of genetic mango pollen, and provides a basis for distinguishing different mango varieties, and further improves the genetic breeding level of mango.

[0046] It is apparent to those skilled in the art that the application is not limited to the details of the foregoing exemplary embodiments, and that the application can be implemented in other specific forms without departing from the spirit or essential characteristics of the application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, and the scope of the application should be defined by the appended claims rather than by the foregoing description, and it is intended to include all changes falling within the meaning and range of equivalents of the claims.

[0047] Furthermore, it should be understood that, although the present specification is described in terms of embodiments, not every implementation embodies an independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A method of preparing a pollen scanning electron microscope sample, characterized by, Specifically comprising the following steps: Step 1, gently take the anther with tweezers, keep the pollen on the anther of stamen to avoid the pollen from scattering, facilitate the sticking table processing; put it into a glass dish, and make a mark; Step 2, place the glass dish containing the pollen under the center of the near-infrared baking lamp at a distance of 20 cm, dry for 90 min, then move the glass dish to the edge, and use the edge temperature to assist the pollen in drying through radiation and its own heat transfer to dry the sample for 30 min; the infrared spectrum range is 0.7 μm-2.5 μm; Step 3, take a clean weighing paper, after the sample is dried to the outside without excess moisture, take out the anther with tweezers, pat the wrist, evenly drop the pollen on the light surface of the weighing paper through slight vibration, and then turn over the sample table with conductive glue evenly stuck on it; Step 4, put the pollen sample table into the ion sputtering instrument one by one, and sputter the alloy on the surface of the sample for 60 s; Step 5, observe the micro-morphology of the pollen sample through scanning electron microscopy, and set the uniform acceleration voltage to 5 kv.

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