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Jun 2011
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Rice Root Hair Phenotypes Imaged by Cryo-SEM
水稻根毛表型的低温扫描电镜成像   

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Abstract

Cryo-scanning electron microscopy (cryo-SEM) was first introduced for scientific use in the 1980s. Since then, cryo-SEM has become a routine technique for studying the surfaces and internal structures of biological samples with a high water content. In contrast to traditional SEM, cryo-SEM requires no sample pretreatment processes; thus, we can obtain the most authentic images of the sample shape and structure. Cryo-SEM has two main steps: cryoprocessing of samples and scanning electron microscopy (SEM) observation. The cryoprocessing step includes preparation of the cooled slushing station, cooling of the preparation chamber, sample preparation, and sputtering. The sample is then transferred to an SEM cold stage for observation. We used cryo-SEM to study rice root hair tissues, but the methods and protocols can be applied to other root systems. This protocol optimizes the two key operation steps of reducing the humidity in the growth chamber and previewing the samples before sputtering and can more quickly obtain high-quality images.

Keywords: Cryo-SEM (冷冻扫描电镜), Rice (水稻), Mutant (突变), Root hair (根毛), Diameter (直径), Density (密度), Cell interior (细胞内部)

Background

Scanning electron microscopy (SEM) refers to the use of an electron beam apparatus and a pattern inspection apparatus to image a sample surface (Yan, 2010). The first SEM came on the market in 1965 when the Cambridge Instrument Company launched a commercial instrument (McMullan, 1995). Specifically, biological samples must be dehydrated before entering the SEM chamber (Echlin, 1971), otherwise water vapor contaminates the electron microscope vacuum system. Before cryopreservation was invented, freeze-drying and critical point drying were two commonly used methods (Sargent, 1986). These pretreatments were inevitably associated with sample distortion, shrinkage, or loss of inner cellular soluble components (Reference 1; Echlin, 1971); additionally, they were time-consuming and laborious.


The water in a biological material generally forms ice crystals when the sample freezes at 0°C or below; however, the water forms a glasslike structure when the material is cooled at a very high cooling rate (Rey, 1960; Binder, 2014; Limmer and Chandler, 2014). In comparison with the crystal structure, this glasslike, amorphous water minimizes damage to the cell structure and mechanical properties (Vega-Gálvez et al., 2008); therefore, cryo-scanning electron microscopy (cryo-SEM) was devised to exploit this advantage.


An exceptional paper presented a detailed and comprehensive description of cryo-SEM development and its application in biology (Read and Jeffree, 1991). In short, cryo-SEM was first performed in 1960 but was not widely known until 1970 (Echlin, 1971). Commercial cryopreparation systems were available for SEMs in the 1980s; henceforth, cryo-SEM technology became routine and was commonly used in biological research (Read and Jeffree, 1991). Additionally, cryo-SEM was widely used in the field of botany for observations of the surface or freeze-etched fractions of roots (Vartanian et al., 1983; Webb and Jackson, 1986; Ryan et al.,1998; Dolan et al., 1994; Foreman and Dolan, 2001; Müller and Schmidt, 2004; Ding et al., 2009; Yi et al., 2010; Zhiming et al., 2011; Huang et al., 2013; Zou et al., 2015; Giri et al., 2018; Wang et al., 2019; Zenone et al., 2020), stems (Echlin, 1971), vessels (Utsumi et al., 1998), leaves (Sargent, 1983), shoot apexes (Kaneko, 1985), glandular trichomes (Kaneko, 1985), stamen hair cells (Kaneko, 1985), stigmas (Kaneko, 1985), petals (Wang et al., 2019), pollen grains (Echlin, 1971; Berger et al., 1998), stomatal pores (Echlin, 1971), berry skins (Brizzolara et al., 2020), seeds (Yu et al., 2014), etc. Moreover, cryo-SEM has been applied to the visualization of root-fungal interactions (Refshauge et al., 2006).


Root hairs are a type of tubular protuberant cell that diverges from specific root epidermal cells (Ishida et al., 2008; Kim and Dolan, 2016). They are capable of increasing root and soil contact areas and improving the efficiency of water and nutrient absorption while providing a place for the plant to interact with soil microorganisms (Larkin et al., 2003).


Without cryo-SEM, it is difficult to obtain favorable results for root hairs because of the large proportion of water inside root hairs. Root hair shape maintenance is dependent on turgor pressure driven by inner water (Mendrinna and Persson, 2015). Essentially, some samples, such as primary roots (Lefebvre, 1985), leaves (Eveling and McCall, 1983; Sargent, 1983), petals (Chen and Meyerowitz, 1999), and stamens (Chen and Meyerowitz, 1999), can remain intact after drying treatment and be photographed by SEM.


Nevertheless, with effective cryo-SEM technology, root hairs inevitably shrink and/or bend during cryo-preparation (Dolan et al., 1994; Czarnota et al., 2003; Cocozza et al., 2008; Zou et al., 2015); therefore, the process of preparing cryo-SEM samples is extremely important. In the following sections, some operations are highlighted and introduced in detail. In the foreseeable future, this technology will be suitable for stamen filament samples and other samples similar to root hair.


Advantages of Cryo-SEM:

  1. It is suitable for tissue samples with a high moisture content.

  2. Samples do not need to be fixed or dehydrated in advance.

  3. It is proficient at revealing details that optical microscopy cannot.

  4. Detailed quantitative parameters of the root hairs can be obtained.

  5. The interior cell structure can be studied.


Disadvantages of Cryo-SEM:

  1. Rice roots must be cultured in an agar plate.

  2. Root sample throughput is fairly low as compared with other methods.

  3. Sample pretreatment requires professional operation skills.

  4. Price of sample pretreatment is relatively high.


Materials and Reagents

  1. 3MTM MicroporeTM Surgical Tape (3M, catalog number: 1533)

  2. Nitrocellulose membranes with 0.8 μm pore size, 25 mm filter diameter (AAWG02500, Millipore, Gemany)

  3. Rice root hair mutant Osbhlh115 seeds (Ding et al., 2009); Oryza sativa L. Xian group (also known as Hsien or Indica) wild-type Kathalath seeds

  4. Sodium hypochlorite (BBI Life Sciences Corporation, China)

  5. 70% ethyl alcohol (BBI Life Sciences Corporation, China)

  6. Murashige and Skoog Basal Medium (Duchefa Biochemie, catalog number: M0221)

  7. Phytagel (Sigma-Aldrich, catalog number: P8169-250G)

  8. Carbon-rich conductive glue (Rave Scientific, catalog number: RS-MN-15-001130)

  9. Gold wafer (70-AU2408, 24 × 0.2 mm, purity 99.99%. Au, Labtech, UK)

  10. Argon gas (99.99% purification, Hangzhou Jingong, China)

  11. Nitrogen gas (99.99% purification, Hangzhou Jingong, China)

  12. Liquid nitrogen (Hangzhou Jingong, China)

Equipment

  1. Horizontal shaker (Beyotime, model: TS-2000A)

  2. Growth chamber (Ningbo-Jiangnan, model: GXM-1008)

  3. Scanning electron microscope (HITACHI, model: S-3000N)

  4. GATAN ALTO 2100 (GATAN, model: ALTO 2100)

  5. HITACHI SEM equipped with a GATAN cryo-SEM preparation system

Software

  1. ImageJ (Version 1.53e, National Institutes of Health, United States, https://imagej.nih.gov/ij)

  2. Office 365 (Microsoft, United States, https://www.office.com/)

Procedure

  1. Rice Root Hair Growth

    1. Sterilize the glume removed-rice seeds with 70% EtOH for 2 min, and remove the disinfectant alcohol. Then, sterilize the seeds with 10% sodium hypochlorite for 30 min on a horizontal shaker. Rinse the seeds thoroughly 5 times with sterile distilled water.

    2. Place the seeds on sterile absorbent paper for drying.

    3. Sow the seeds onto 0.8% phytagel half-strength MS medium (pH = 5.8).

      Note: To avoid too much moisture attached to the root hairs, it is highly recommended to seal the medium plates with 3M Micropore surgical tape.

    4. Place the medium plate vertically in the growth chamber under a temperature regime of 30°C/22°C (day/night) with 50% or lower humidity and a 12 h photoperiod (15,000 lux).

      Note: Lowering the humidity is an effective way to reduce the moisture in the plate.

    5. Use three-day-old rice roots after germination for subsequent sample treatment.


  2. Preparation for Cryo-SEM

    1. Install the SEM stage cold module (Figures 1; see Video 1).



      Figure 1. SEM stage cold module installation. Dovetail and fit the module with a thermometer. Push the copper shaft and copper shaft sleeve together to make an interference fit connection. Ensure that the real-time module temperatures are displayed on the keypad.


      Video 1. SEM stage cold module installation


    2. Check the Alto 2100 keypad (or control panel) (Figure 2).



      Figure 2. The keypad at the very beginning of the start-up. The keypad allows control of the pump and the system’s valves, sputter coater, lighting, and heater. All temperature and pressure readings are instantly available and easy to observe on the keypad.


    3. Prep chamber precooling (Figure 3, see Video 2)



      Figure 3. The prep-chamber before adding liquid nitrogen. The red circles indicate the inlet and outlet for liquid nitrogen. The red ellipse indicates the pressure indicator LED display.


      Video 2. Pour the liquid nitrogen into the prep chamber inlet

    4. Precool the SEM cold stage module

      Pour liquid nitrogen into the open topped dewar where the pipeline is immersed (see Video 3). The pipeline is filled with high-purity nitrogen gas. The cooled gas in the pipeline cools down the SEM stage cold module below -180°C.


      Video 3. Pour liquid nitrogen into the open topped dewar

      Note: Ventilate the cryo-pipeline for at least 5 min before running the cryo-SEM system. A uniform flow rate of nitrogen in the pipeline is necessary for image quality.


  3. Produce slush nitrogen

    1. Pour the liquid nitrogen into the slush chamber and close the lid (see Video 4).

      Note: Wear waterproof cryogloves!


      Video 4. Preparation of slush nitrogen

    2. Press the SLUSH/VENT button on the Alto control panel to activate slush chamber pumping (see Video 4).

    3. Allow the liquid nitrogen to boil until it solidifies. This step takes approximately two minutes.

    4. Press the SLUSH/VENT button again to stop pumping and vent the air into the chamber.

    5. Remove the lid before use.


  4. Root Sample Acquisition

    1. Cut a 1-2 cm length of wild type or mutant straight growing seminal roots for use.

    2. Add a drop of carbon-rich conductive glue to the aluminum stub of the specimen holder (see Video 5).


      Video 5. The conductive glue was applied to the aluminum stub

    3. Place a piece of moist nitrocellulose paper, immersed in distilled water in advance, on the glue (see Video 6).


      Video 6. Putting the nitrocellulose paper onto the sample stage and applying gel

    4. Gently clamp the cut-off root end with tweezers and transfer the root onto nitrocellulose paper. Place the root as horizontally as possible (Figure 4).



      Figure 4. Clamping the rice root onto the nitrocellulose paper. First, apply conductive glue to the aluminum stub. Then, place the nitrocellulose paper on the gel. After that, spread the conductive glue to the near end of the rod. Last, stick the root cut end on the glue and lay the root flat on the nitrocellulose paper.


    5. Immerse the trimmed root end in the glue (Figure 4).


  5. Root Sample Repreparation

    1. Stick together the specimen holder with a root sample on its stub and the sample holder integrated with the vacuum transfer device (VTD) (see Video 7).

    2. Buckle the sample holder (with the VTD) to the slush chamber until the VTD sits on the O-ring in the recess located on the top of the slushing chamber (see Video 7).

    3. Slide the rod in the VTD down (see Video 7).

    4. Grip the sample holder in the air above the nitrogen pool for 10 s to quickly freeze the root hair (see Video 7).

    5. Plunge the sample holder into the slushy nitrogen to rapidly cool the sample (see Video 7).

      Note: Ensure that only the sample holder is immersed in the nitrogen when slushing, otherwise the cold rod freezes the VTD’s O-ring. This has a negative influence on the subsequent push-pull operation and/or the gas tightness.


      Video 7. The sample immersed into the nitrogen slush

    6. Press the SLUSH/VENT button to activate slush chamber pumping.

    7. Pull the VTD rod upward and fully retract the sample holder into VTD before the nitrogen slush completely solidifies (see Video 8).


      Video 8. The sample waiting to leave the slushing station

    8. Push the trapdoor valve (see Video 9) (The sample is now held in the VTD under a vacuum and ready for transfer into the prep chamber).


      Video 9. The sample leaving the slushing station


    9. Press the SLUSH/VENT button once again to vent the slush chamber.

    10. Transfer and place the VTD in the GATE VALVE airlock (see Video 10).


      Video 10. Pump the Gate Value airlock vacuum

    11. Press the LOAD PUMP button on the Alto control panel to vacuum the prep chamber (Figure 2).

    12. Wait until six bars of indicator light illuminate, which is indicated by the red ellipse in Figure 3.

    13. Fully open the valve, push the rod, and insert the specimen holder into the dovetail stage in the prep chamber (Figures 5-6; see Video 11).



      Figure 5. The dovetail stage in the prep-chamber. The dovetail stage is convenient for the angled sides of the sample holders and is fitted with a thermometer and a heater. There is a stopper to allow the sample holder to be transferred to exactly the same position. A spring clip secures the holder and prevents vibration.



      Figure 6. The rice root sample before sublimating and sputtering. The white circle indicates that the sample is waiting to be sublimated and sputtered on the stage.


      Video 11. Transfer the sample to the prep chamber (The VTD and prep chamber valves are opened simultaneously)

    14. Set the sublimation temperature of the prep-chamber stage to -95°C (Figure 7).



      Figure 7. The sublimation temperature settings. By pressing the SET TEMPERATURE buttons, set the sublimation temperature to -95°C.


    15. Press the HEAT button to start warm-up (see Video 12) (For rice root hairs, maintaining at -95°C for 300 s is optimal. The sublimation time mainly depends on the humidity of the root culture environment).


      Video 12. The sublimation temperature keeps rising

    16. Press the HEAT button again to stop heating and recover the temperature to ≤ -140°C.

    17. Set the gold sputter time and press the SPUTTER button on the control panel (120 s is sufficient for root hairs) (Figure 8).

      Note: If the prep chamber is not equipped with a microscope device, samples can be transferred to the SEM chamber for rough viewing before sputtering. This step is extremely important for obtaining favorable samples and high-quality images.



      Figure 8. The sputter parameter display. The default setting of the sputter electric current is 10 mA. Before argon sputtering, the stage temperature should be cooled to ≤-140°C.


    18. The argon inlet solenoid closes automatically when the time is up (Figure 9; see Video 13).



      Figure 9. The rice root being sputtered. The rice root sample should be placed under the coating head during sputtering.


      Video 13. The sample is being sputtered

  6. SEM Microscopy

    1. Open the ball valve between the preparation chamber and the SEM chamber when the indicator LED light turns green (Figures 10-11).



      Figure 10. The ball valve LED is red. In this situation, the mechanical interlock cannot be opened.



      Figure 11. The ball valve LED is green. When the vacuum in the prep chamber and the SEM equilibrates, the LED turns green, and the mechanical interlock can be opened.


    2. Transfer the specimen holder to the SEM cold stage module (see Video 14).


      Video 14. Transfer the sample from the prep chamber to the SEM

    3. Rotate the rod and separate the specimen holder from the rod.

    4. Retract the rod back into the prep chamber and close the ball valve. SEM imaging has now started.

    5. Open the software and click the HV button to start the preview and shooting mode.

    6. Set the SEM acceleration voltage (6 kV or 15 kV).

    7. Adjust the SEM sample stage position by rotating different control knobs for the SEM standard stage (Figure 12).

    8. Rotate the rotary functional knobs on the HITACHI control unit by adjusting MAGNIFICATION, CONTRAST, BRIGHTNESS, and FOCUS (Figure 13).



      Figure 12. The standard stage of the SEM. The T control knob tilts the specimen (-20 to 90°); the R control knob rotates the specimen (±360°); the X control knob moves the specimen in the longitudinal direction; the Y control knob moves the specimen in the lateral direction; the Z control knob moves the specimen in the vertical direction.



      Figure 13. Appearance of the SEM display unit. The rotary knob unit is shown in the yellow rectangle. The control button layout on the unit is embedded in the image.


    9. Click the mouse to open or close dialog windows and run the settings. Input characters and comments with the keyboard.

    10. Switch to different sample parts by moving the mouse wheel.

    11. Click the H.R. Capture button to scan the sample. The scanning speed is optional.

      Note: The slower the scanning speed, the higher the resolution of the image. However, a slow scanning speed occasionally causes image deformation.

    12. Click the SAVE button to save and name the acquired image in the target folder.

    13. Click the H.V. button again to turn off the SEM filament when imaging is finished.

    14. Open the BALL VALVE, and remove the specimen holder from the Cold Stage Module located by the rod.

    15. Haul the rod backwards and insert the holder into the dovetail stage in the prep chamber.

    16. Close the BALL VALVE.

    17. Haul the rod backwards to the end and shut the airlock valve.

    18. Push the LOAD PUMP button on the Alto control panel twice and vent the airlock to remove the VTD.

    19. Place the VTD on the slushing chamber and engage the trapdoor valve onto the control lever.

Data analysis

Root hair parameters were obtained from the cryo-SEM images (Figure 14). The root hairs from the region of 2-3 mm from the root apex were used for the statistics and analysis of the root hair parameters. According to the results, the root hair length in the Osbhlh115 mutant was much shorter than that in the wild type (Table 1). The difference in root hair diameter between the wild type and mutant was insignificant. Although the angles between root hairs and seminal roots of the wild type and mutant were not different, cryo-SEM provided an important tool for the study of the root hair angle parameter (Table 1).



Figure 14. Root hair phenotype analyzed by cryo-SEM. A-B. Kathalath (wild type) root hair phenotype. A is 40× magnification and B is 400× magnification; C-D. Osbhlh115 mutant root hair phenotype. C is 40× magnification and D is 400× magnification.


Table 1. Quantitative parameters of the root hairs*

Sample Root hair length (µm) Root hair diameter (µm) Angle between root hair and seminal root (°)
Wild type 177.92±11.00 6.26±0.03 79.67±4.42
Mutant 26.93±1.56 6.34±0.14 80.17±5.15

*All data were obtained according to the reference instructions (Tajima and Kato, 2013)


In addition to imaging root hairs, cryo-SEM can be used to image root cell internal structures (Figure 15). After the water in the epidermal and cortical cells is removed by sublimation, the reticulate structure and layered structure, respectively, at 2,000× magnification are shown (Figure 15B). Similar cytoplasmic lamellar and reticulate structures were found in the drupelet mesocarp cells of red raspberry (Rubus indaeus L.) (Williamson and Duncan, 1989). Conventional paraffin and resin sections could not show the true structures of the cytoplasm. When resin or paraffin replaced the water in the tissue, it likely took away some of the proteins or other components in the cytoplasm. Cryo-SEM can eliminate water through sublimation, retaining the cytoplasmic contents to the greatest extent. It was reported many years ago that electron microscope freeze-fixation was better than chemical fixation (Walther and Müller, 1999).



Figure 15. Cytoplasmic contents analyzed by cryo-SEM. A. Kathalath root transverse fracture cytoplasmic structure at 180× magnification. B. Enlarged view of the red rectangular zone in B at 2,000× magnification. The red circle in B indicates a reticulate structure; the red square in B indicates a lamellar structure.


Nitrogen sludge may transform into nitrogen solids during the root hair freezing process. The root hair tubular protuberant parts were broken occasionally when the specimen holder was retracted from the slush chamber. In Figure 16, a variety of broken root hair holes are visible (Figure 16A). Root hairs exhibit a large vacuole in the protruding part (Preuss et al., 2004). After the sublimation finished, the entire vacuole was evaporated to dryness and an empty cavity was left (Figure 16B). Some of the residual particles in the cavity most likely represent cytoskeletal proteins (Walther and Müller, 1999). In conclusion, there are reasons to believe that the reticulate and layered structures displayed in Figure 15B have different functions in the formation of cell morphology, organelle positioning, signal transduction, and material transportation. Note that only a small portion of the information displayed by cryo-SEM was utilized and further exploration is needed.



Figure 16. Broken root hairs left holes. A-B. Kathalath (wild type) root hair phenotype. A is 400× magnification and B is 7,500× magnification.

Acknowledgments

This project was supported by Zhejiang Provincial Natural Science Foundation (LY19C020002), the National Science Foundation of China NSFC (31200913), the Entrepreneurship and Innovation Project for the Overseas Returnees (or Teams) in Hangzhou (4105C5062000611), China Scholarship Council (201709645003) and National Undergraduate Innovation and Entrepreneurship Training Program (1085C5212030510). This protocol was adapted from Yu et al. (2013). We give great thanks to Mr. Hanmin Chen at Zhejiang University for his technical support. Our personal heartfelt appreciation goes to Dr. Zhiming Yu’s late advisor Professor Dr. Ping Wu at Zhejiang University. The protocol presented here was developed from a previous publication (Yu et al., 2011).

Competing interests

The authors declare no competing financial interests.

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简介

[摘要] Cryo-扫描电子显微镜(低温SEM)首次介绍了在80年代科学使用。此后,冷冻-SEM已成为研究常规技术的表面和内部结构与生物样品一水高含量。与传统的SEM相比,cryo -SEM不需要样品预处理过程。因此,我们可以得到最真实的图像的的样品的形状和结构。低温SEM有两个主要步骤:样品的冷冻处理和扫描电子显微镜(SEM)观察。所述cryoprocessing步骤包括准备所述冷却站抗蚀,冷却的所述制备腔室,样品制备,和溅射。然后将样品转移到一个SEM冷阶段观察。我们使用cryo-SEM研究了水稻根毛组织,但是该方法和协议可以应用于其他根系。该协议优化了降低生长室中的湿度和在溅射前​​预览样品的两个关键操作步骤,并且可以更快地获得高质量的图像。



[Backgrou ND ]扫描电子显微镜(SEM)是指使用电子束装置的和图案检查装置的图像的样品表面(严,2010)。1965年,当剑桥仪器公司推出了商用仪器时,第一台SEM上市(McMullan,1995年)。具体而言,生物样品在进入SEM室之前必须进行脱水(Echlin,1971),否则水蒸气会污染电子显微镜真空系统。在发明冷冻保存之前,冷冻干燥和临界点干燥是两种常用的方法(Sargent,1986)。这些预处理不可避免地与样品的变形,收缩或内部细胞可溶性成分的损失有关(参考文献1;Echlin,1971)。此外,它们既费时又费力。

当样品在0°C或更低的温度下冻结时,生物材料中的水通常会形成冰晶。ħ H但是,在水中形成当该材料在非常高的冷却速度冷却玻璃状结构(雷伊,1960;粘合剂,2014; Limmer和Chandler,2014)。与晶体结构相比,这种玻璃状无定形水将对细胞结构和机械性能的损害降到最低(Vega- Gálvez等,2008 )。吨herefore,低温扫描电子显微镜(低温SEM)被设计成利用此优点。

一篇出色的论文详细介绍了冷冻-SEM的发展及其在生物学中的应用(Read和Jeffree,1991)。简而言之,cryo-SEM于1960年首次进行,但直到1970年才广为人知(Echlin,1971)。1980年代,用于SEM的商业低温修复系统问世。此后,低温SEM技术成为常规技术,并在生物学研究中广泛使用(Read和Jeffree,1991)。另外,cryo-SEM在植物学领域被广泛用于观察根的表面或冷冻蚀刻的部分(Vartanian等,1983; Webb and Jackson,1986; Ryan等,1998; Dolan等。,1994; Foreman和Dolan,2001;Müller和Schmidt,2004;Ding等,2009;Yi等,2010;Zhiming等,2011;Huang等,2013;Zou等,2015; Chen等,2011 ;Zhang等,2009。Giri等人,2018; Wang等人,2019 ; Zenone等人,2020 ),茎(Echlin,1971),容器(Utsumi等人,1998),叶片(Sargent,1983),芽顶(Kaneko) ,1985),腺毛(Kaneko,1985),雄蕊毛细胞(Kaneko,1985),柱头(Kaneko,1985),花瓣(Wang等,2019 ),花粉粒(Echlin,1971 ; Berger等,, (1998年),气孔(Echlin,1971年),浆果皮(Brizzolara等人,2020年),种子(Yu等人,2014年)等。此外,cryo-SEM已用于可视化根真菌相互作用。(Refshauge等,2006)。

根毛是一种与特定的根表皮细胞不同的管状突起细胞(Ishida等,2008; Kim and Dolan,2016)。它们能够增加根与土壤的接触面积,提高水分和养分吸收的效率,同时为植物提供了与土壤微生物相互作用的场所(Larkin等,2003)。

没有冷冻SEM,由于根毛中水的比例很大,因此很难获得对根毛有利的结果。保持根毛的形状取决于内部水驱动的膨胀压力(Mendrinna和Persson ,2015)。从本质上讲,一些样品,例如初生根(Lefebvre,1985 ),叶片(Eveling和McCall,1983; Sargent,1983),花瓣(Chen和Meyerowitz,1999)和雄蕊(Chen和Meyerowitz,1999)都可以保持完整。干燥处理后,用SEM拍照。

然而,采用有效的低温SEM技术,在低温制备过程中根毛不可避免地会收缩和/或弯曲(Dolan等,1994;Czarnota等,2003;Cocozza等,2008; Zou等,2015)。; 吨herefore,制备低温SEM样品的过程是非常重要的。在以下各节中,将突出显示并详细介绍一些操作。在可预见的将来,该技术将适用于雄蕊细丝样品和其他类似于根毛的样品。



低温扫描电镜的优点:

它适用于与组织样品一个高水分含量。
样品无需预先固定或脱水。
它擅长揭示光学显微镜无法发现的细节。
可以获得根毛的详细定量参数。
可以研究内部细胞结构。


低温SEM的缺点:

稻根必须在培养的琼脂板。
根样品通量是相当低的比较与其他方法。
样品预处理需要专业的操作技能。
样品预处理的价格相对较高。

关键字:冷冻扫描电镜, 水稻, 突变, 根毛, 直径, 密度, 细胞内部

材料和试剂

3M TM Micropore TM手术胶带(3M,目录号:1533)
用0.8硝酸纤维素膜微米孔径大小为25mm滤波器的直径(AAWG02500,Millipore公司,Gemany )
水稻根毛突变体Osbhlh115种子(Ding等,2009 );水稻冼组(也称为县或籼)野生-类型Kathalath种子
次氯酸钠(BBI生命科学公司,中国)
70%乙醇(BBI生命科学公司,中国)
Murashige和Skoog基础培养基(Duchefa Biochemie ,目录号:M0221)
Phytagel(Sigma-Aldrich,目录号:P8169-250G)
富碳导电胶(Rave Scientific,目录号:RS-MN-15-001130)
金晶片(70-AU2408,24 × 0.2 mm,纯度99.99%。Au ,Labtech,UK)
氩气(99.99%的纯度,中国杭州金宫)
氮气(99.99%的纯度,中国杭州金宫)
液氮(中国杭州金宫)
设备

ħ orizontal摇床(碧云天,模型:TS-2000A)
生长室(宁波-江南,型号:GXM-1008)
扫描电子显微镜(日立,型号:S-3000N)
GATAN ALTO 2100(GATAN,型号:ALTO 2100)
HITACHI SEM配备有一个GATAN低温SEM制备系统
软件

ImageJ(版本1.53e,美国国立卫生研究院,https: //imagej.nih.gov/ij )
Office 365(Microsoft,美国,https://www.office.com/)
程序

水稻根毛生长
用70%的乙醇将去除了颖花的大米种子消毒2分钟,然后除去消毒剂酒精。然后,消毒用10%次氯酸钠的种子上30分钟一个水平振荡器。用无菌蒸馏水彻底冲洗种子5次。
将种子放在无菌吸水纸上干燥。
播种到0.8%phytagel半强度MS培养基(pH = 5.8)上。
注意:为避免过多的水分附着在根毛上,强烈建议使用3M Micropore外科手术胶带密封中板。

将培养基板垂直放置在温度为30°C / 22°C(白天/夜晚),湿度为50%或更低,光周期为12小时(15,000 lux)的生长室中。
注意:降低湿度是减少印版中水分的有效方法。

发芽后使用三天大的稻根进行后续样品处理。
低温SEM的准备
安装SEM载物台冷模块(图1;请参见视频1 )。
图1. SEM台冷模块的安装。燕尾榫并在模块中安装温度计。将铜轴和铜轴套推到一起以形成过盈配合的连接。确保实时模块温度显示在键盘上。

视频1. SEM载物台冷模块的安装

检查Alto 2100键盘(或控制面板)(图2 )。
图2.刚开始时的键盘。该键盘允许控制所述泵和所述系统的阀,溅射镀膜机,照明,和加热器。所有温度和压力读数均可立即获得,并且易于在键盘上观察。

准备室预冷(图3,请参见视频2 )
图3.添加液氮之前的预燃室。红色圆圈表示液氮的入口和出口。红色的椭圆形表示压力指示器的LED显示屏。

              视频2.倒入液氮中,以预备室入口

预冷SEM冷台模块
倾液氮到顶部开口杜瓦瓶,其中管道被浸没(小号EE视频3)。管道中充满了高纯氮气。管道中的冷却气体将SEM级冷模块冷却至-180 °C以下。

视频3.将液氮倒入开顶的杜瓦瓶中

注意:在运行cryo-SEM系统之前,请对低温管道通风至少5分钟。管道中氮气的均匀流速对于图像质量是必不可少的。

产生氮泥
将液氮倒入冲洗池中,然后关闭盖子(请参见视频4 )。
注:耐磨防水CR ÿ ogloves !

视频4 。溶解氮的制备

按下Alto控制面板上的SLUSH / VENT按钮以激活污泥室泵送(请参见视频4 )。
让液氮沸腾直到固化。此步骤大约需要两分钟。
再次按下SLUSH / VENT按钮,以停止泵送并将空气排入腔室。
使用前请取下盖子。
根样本采集
切开1-2厘米长的野生型或突变的直生精根以供使用。
将一滴富含碳的导电胶添加到样品架的铝制短管中(请参见视频5 )。
录像5 。将导电胶涂到铝棒上

将一张预先浸入蒸馏水中的潮湿硝化纤维素纸放在胶水上(请参见视频6 )。
录像6 。将硝酸纤维素纸放在样品台上并涂上凝胶

轻轻夹住切-断根端与镊子和根转移到硝酸纤维素纸上。将根尽可能水平放置(图4 )。
图4 。将米根夹在硝酸纤维素纸上。首先,将导电胶涂到铝桩上。然后,将硝酸纤维素纸放在凝胶上。之后,将导电胶涂抹到杆的近端。最后,将切好的根部末端粘在胶水上,然后将根部平放在硝酸纤维素纸上。

将修剪后的根端浸入胶中(图4 )。
根样品准备
将带有根样品的样品支架和存根以及与真空传输设备(VTD)集成在一起的样品支架粘在一起(请参见视频7 )。
将样品架(与VTD一起)扣紧到冲洗池上,直到VTD固定在冲洗池顶部凹槽中的O形圈上(请参见视频7 )。
向下滑动VTD中的杆(请参见视频7 )。
在氮气池上方的空气中将样品架夹住10秒钟,以快速冻结根毛(请参见视频7 )。
将样品架浸入泥泞的氮气中,以快速冷却样品(请参见视频7 )。
注意:确保当抗蚀只有样本保持器浸没在氮,Ó therwise冷杆冻结VTD的O形环。这对随后的推挽操作和/或气密性具有负面影响。

视频7 。样品浸入氮气中

按下SLUSH / VENT按钮,以启动污泥室泵送。
向上拉动VTD杆,将样品架完全缩回到VTD中,直到氮泥完全固化(请参见视频8 )。
录像8 。等待样品到LEAV Ë的抗蚀站

推动活板门阀(见视频9 )(样品现在在VTD保持下一个真空,并准备转移到预备室)。
视频9 。样品离开融泥台

再次按下SLUSH / VENT按钮以排空泥浆室。
转移VTD并将其放置在GATE VALVE气闸中(请参见视频10 )。
录像10 。抽出Gate Value气闸真空

按下Alto控制面板上的“ LOAD PUMP”(加载泵)按钮以抽真空准备室(图2 )。
等到指示器六个条光照射,这是由所指示的红色椭圆在图3 。
完全打开阀,推动杆,然后将试样保持器进入燕尾阶段的预备室(图5- 6 ;请参见视频11 )。
图5.预备室中的燕尾榫阶段。燕尾阶段方便了样品容器的成角度的边,并配有一温度计和加热器。每个挡块都有一个挡块,可将样品架移至完全相同的位置。弹簧夹固定支架并防止振动。

图6.升华和溅射前的稻根样品。白色圆圈表示样品正在等待升华并溅射在平台上。

录像11 。将样品转移到制备室(VTD和制备室阀门同时打开)

将预燃室的升华温度设置为-95 °C(图7 )。
图7.升华温度设置。通过按SET TEMPERATURE按钮,将升华温度设置为-95 °C 。

按下加热按钮,开始预热(见视频12 )(对于水稻根毛,在-95℃下保持300秒是最佳的。升华时间主要取决于湿度的根培养环境)。
视频12 。升华温度持续上升

再次按HEAT(加热)按钮停止加热,并将温度恢复到≤- 140°C。
设置金溅射时间,并按下控制面板上的按钮SPUTTER(120秒是足以对根毛)(图8 )。
注意:如果准备室未配备显微镜设备,则可以在溅射之前将样品转移到SEM室进行粗略观察。这一步对于获得良好的样本和高质量图像非常重要。

图8.溅射参数显示。溅射电流的默认设置为10 mA。在进行氩气溅射之前,应将工作台温度冷却至≤- 140 °C 。

的氩气入口螺线管关闭时,自动的时间到了(图9;参见视频13 )。
图9.溅起的稻草根。溅射时,稻根样品应置于包衣头下方。

视频13 。样品被溅射

扫描电镜
当指示灯LED指示灯变为绿色时,打开制备室和SEM室之间的球阀(图10-11 )。
图10.球阀LED为红色。在这种情况下,机械互锁无法打开。

图11.球阀LED为绿色。当在真空中预备室和SEM达到平衡时,LED变绿,并且机械互锁可以被打开。

将样品架移至SEM冷台模块(请参见视频14 )。
视频14 。从转移样品的制备室的SEM

旋转杆,并将样品支架与杆分离。
将杆缩回到准备室并关闭球阀。SEM成像公顷š现在开始。
打开软件,点击HV按钮启动的预览和拍摄模式。
设置SEM加速电压(6 kV或15 kV)。
通过旋转SEM标准载物台的不同控制旋钮来调整SEM样品载物台的位置(图12 )。
通过调节放大,对比度,亮度和聚焦来旋转HITACHI控制单元上的旋转功能旋钮(图13 )。
图12. SEM的标准阶段。T控制旋钮使样品倾斜(-20至90°);R控制旋钮旋转样品(±360°); X控制旋钮可沿纵向移动样本;Y控制旋钮在横向方向上移动样本;Z控制旋钮可沿垂直方向移动样本。

图13. SEM显示单元的外观。旋钮单元以黄色矩形显示。设备上的控制按钮布局已嵌入到图像中。

单击鼠标以打开或关闭对话框窗口并运行设置。用键盘输入字符和注释。
通过移动鼠标滚轮切换到不同的样品部件。
单击“ HR捕获”按钮以扫描样品。扫描速度是可选的。
注意:扫描速度越慢,图像的分辨率越高。但是,较低的扫描速度有时会导致图像变形。

单击“保存”按钮以将获取的图像保存并命名为目标文件夹。
再次单击该按钮,HV关闭的,当成像完成SEM长丝。
打开球阀,然后从位于杆上的冷台模块上取下样品架。
向后拖拉杆,然后将固定器插入准备室的燕尾槽台中。
关闭球阀。
将杆向后拖到底,然后关闭气闸阀。
按下Alto控制面板上的LOAD PUMP按钮两次,并打开气闸以卸下VTD。
将VTD放在冲洗室上,然后将活板阀接合到控制杆上。
数据一nalysis

从cryo-SEM图像获得根毛参数(图14 )。来自根尖2-3mm区域的根毛用于统计和分析根毛参数。根据该结果,Osbhlh115突变体的根毛长度比野生型的根毛长度短得多(表1 )。的在差的野生型和突变体之间根毛直径不显着。尽管野生型和突变型的根毛与精根的夹角没有区别,但cryo-SEM为研究根毛夹角参数提供了重要的工具(表1 )。

图14.通过冷冻-SEM分析的根毛表型。AB。Kathalath (野生型)根毛表型。A是40 ×放大倍率,B是400 ×放大倍率;光盘。Osbhlh115突变根毛表型。C为40 ×放大倍率,D为400 ×放大倍率。

表1.根毛的定量参数*

*所有数据均根据参考说明获得(Tajima和Kato,2013年)

除了对根毛进行成像之外,cryo-SEM还可以用于对根细胞内部结构进行成像(图15 )。在表皮和皮层细胞的水被升华,除去的后交联网状结构和层状结构,分别,在2000 ×放大倍数示出(图15B )。在红树莓(Rubus indaeus L.)的果核中果皮细胞中发现了类似的胞质层状和网状结构(Williamson and Duncan,1989)。常规石蜡和树脂切片无法显示细胞质的真实结构。当树脂或石蜡代替组织中的水时,它可能会带走细胞质中的某些蛋白质或其他成分。低温扫描电镜可以通过升华消除水分,最大程度地保留细胞质含量。它被报道了许多年前的那个电子显微镜冷冻固定比化学固定(瓦尔特和米勒,1999年)更好。

图15.通过cryo-SEM分析的细胞质含量。A. Kathalath根反节骨折细胞质结构在180 ×放大倍数。B. B中红色矩形区域的放大图,放大倍数为2,000 × 。B中的红色圆圈表示网状结构。B中的红色正方形表示层状结构。

根毛冻结过程中,氮泥可能会转化为氮固体。当标本支架从泥浆室缩回时,根发管状突起部分偶尔会断裂。在图16中,可见各种断裂的根毛孔(图16A)。根毛在突出部分表现出较大的液泡(Preuss等,2004)。升华完成后,将整个液泡蒸发至干,并留出空腔(图16B)。空腔中的一些残留颗粒很可能代表细胞骨架蛋白(Walther和Müller,1999)。总之,有理由相信,图15B中显示的网状和分层结构在细胞形态的形成,细胞器定位,信号转导和物质运输中具有不同的功能。请注意,只有一小部分通过cryo-SEM显示的信息被利用,并且需要进一步的探索。

图16.残破的根毛留有小孔。AB。Kathalath (野生型)根毛表型。A为400 ×放大倍率,B为7500 ×放大倍率。

致谢

该项目得到浙江省自然科学基金(LY19C020002),国家自然科学基金委员会(31200913),杭州市海外归国人员(或团队)创业与创新项目(4105C5062000611),中国国家留学基金委(201709645003)的资助以及国家本科生创新创业培训计划(1085C5212030510)。该协议改编自Yu et al。,(2013)。w ^ ê给予十分感谢先生汉民陈在浙江大学为他提供技术支持。我们衷心感谢浙江大学于志明博士已故的顾问吴萍教授。

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引用:Yan, H., Wang, Y., Zhang, J., Cui, X., Wu, J., Zhou, J., Chen, Y., Lu, J., Guo, R., Ou, M., Lai, H. and Yu, Z. (2021). Rice Root Hair Phenotypes Imaged by Cryo-SEM. Bio-protocol 11(11): e4037. DOI: 10.21769/BioProtoc.4037.
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