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Nematode Epicuticle Visualisation by PeakForce Tapping Atomic Force Microscopy
通过峰值力轻敲原子力显微镜观察线虫上表皮   

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Abstract

The free-living soil nematode Caenorhabditis elegans has become an iconic experimental model animal in biology. This transparent animal can be easily imaged using optical microscopy to visualise its organs, tissues, single cells and subcellular events. The epicuticle of C. elegans nematodes has been studied at nanoscale using transmission and scanning (SEM) electron microscopies. As a result, imaging artefacts can appear due to embedding the worms into resins or coating the worms with a conductive gold layer. In addition, fixation and contrasting may also damage the cuticle. Conventional tapping mode atomic force microscopy (AFM) can be applied to image the cuticle of the dried nematodes in air, however this approach also suffers from imaging defects. Ideally, the nematodes should be imaged under conditions resembling their natural environment. Recently, we reported the use of PeakForce Tapping AFM mode for the successful visualisation and numerical characterisation of C. elegans nematode cuticle both in air and in liquid (Fakhrullina et al., 2017). We imaged the principal nematode surface structures and characterised mechanical properties of the cuticle. This protocol provides the detailed description of AFM imaging of liquid-immersed C. elegans nematodes using PeakForce Tapping atomic force microscopy.

Keywords: Atomic force microscopy(原子力显微镜检查), Nematodes(线虫), Caenorhabditis elegans(秀丽隐杆线虫), Cuticle(角质层), PeakForce Tapping(峰值力轻敲), Layer-by-Layer assembly(层层组装)

Background

Nematodes, both free-living and parasitic, have been extensively studied due to their remarkable biology and effects on agriculture and human well-being. Apparently, the most studied and famous species among nematodes is a free-living soil nematode Caenorhabditis elegans (Sterken et al., 2015). This round worm has been successfully employed as a versatile model organism in a number of investigations (Fire et al., 1998; Kenyon, 2010; Swierczek et al., 2011; O’Reilly et al., 2014; Stroustrup et al., 2016), securing eventually a Nobel prize for Sidney Brenner, John Sulston and Robert Horvitz in 2002. C. elegans nematode is a tiny (~1 mm long) transparent animal which can be easily visualised using an optical microscope. Its cuticle, though thin and transparent, serves as a principal protective barrier between the worm and its habitat. Cuticle is an important marker of disease, when pathogenic bacteria colonize it, leading to the death of the infected animal. In addition, the structure of the cuticle may indicate the ageing in the nematodes. From the human health care and industrial point of view, monitoring of the cuticle of nematodes might be helpful in elucidation of novel antinematode chemicals affecting the cuticle. As a result, nanoscale imaging of the nematode epicuticle in its natural liquid environments may open new avenues in biomedical research.

Previously, the cuticle of nematodes (mostly employing C. elegans as a model organism) has been studied using electron microscopy, both transmission and scanning. Unfortunately, electron microscopy does not allow imaging the worms in liquid, which is their native environment. Sample preparation for electron microscopy requires fixation, dehydration, contrast staining or surface sputtering, and also resin embedding for ultrathin slicing. As a result, nematode cuticle may exhibit secondary artifacts preventing from evaluation of native surface structure and mechanical characterisation. Atomic force microscopy has been established as a potent tool in imaging biological samples in situ, including imaging live cells in liquid media (Beaussart et al., 2015). Recently, the application of tapping mode AFM to visualise C. elegans nematodes in air has been reported (Allen et al., 2015). Imaging in air suffered from the same drawback as scanning electron microscopy, for example the images demonstrated the typical shrunk and collapsed cuticle surfaces apparently caused by dehydration and air imaging. We envisaged a different technique, based on PeakForce Tapping AFM mode (Alsteens et al., 2012) for successful visualisation of C. elegans nematode cuticle in water (Fakhrullina et al., 2017). Here we report a detailed protocol for this technique.

Materials and Reagents

Note: All chemicals were purchased from Sigma-Aldrich unless noted otherwise.

  1. Pipette tips
  2. Petri dishes
  3. Dust-free Nexterion glass slides (Schott)
  4. Wild type C. elegans (N2 Bristol) nematodes
  5. Escherichia coli OP50 bacteria
  6. Poly(allylamine hydrochloride) (PAH, molecular weight ~17,5 kDa) (Sigma-Aldrich, catalog number: 283215 )
  7. Poly(sodium 4-styrenesulfonate) (PSS, molecular weight ~70 kDa) (Sigma-Aldrich, catalog number: 243051 )
  8. Nematode growth media (NGM) (bioWORLD, catalog number: 30620040-1 )
  9. Nematode lysis solution (aqueous 2% NaOCl/0.45 M NaOH)
  10. Sodium hypochlorite (NaClO) (Sigma-Aldrich, catalog number: 425044 )
  11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  12. Levamisole hydrochloride (Sigma-Aldrich, catalog number: L9756 )
  13. Glutaraldehyde (25% aqueous) (Sigma-Aldrich, catalog number: G5882 )
  14. Ultrapure (type 1) water purified by Simplicity (Millipore) water purification system
  15. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: NIST200B )
  16. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 71649 )
  17. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  18. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: 793612 )
  19. Aqueous M9 buffer (see Recipes)

Equipment

  1. Pipettes (mechanical Eppendorf or Gilson pipettes)
  2. Eppendorf Scientific Excella E24 temperature-controlled benchtop shaker (Eppendorf, New BrunswickTM, model: Excella® E24 )
  3. Biosan V-1 plus vortex (Biosan, model: V-1 plus )
  4. Nikon SMZ 745 T stereomicroscope (Nikon, model: SMZ745T )
  5. Dimension FastScan Bio atomic force microscope (Bruker, model: Dimension FastScan ) equipped with a liquid submergible scanner
    Note: Do not use air scanners for scanning in liquid, this may damage the instrument.
  6. Biosan Microspin 12 centrifuge (Biosan, model: Microspin 12 )
  7. ScanAsyst-Fluid probes (nominal length 70 µm, tip radius 20 nm, spring constant of 0.7 N m-1) (Bruker, model: SCANASYST-FLUID )
    Note: Other AFM probes from Bruker or other producers approved for using in PeakForce Tapping mode having characteristics similar to ScanAsyst-Fluid probes can also be used.

Software

  1. NanoScope AFM operating software (Bruker Corporation)
  2. NanoScope Analysis v.1.7. software (Bruker Corporation)

Procedure

  1. Fabrication of layer-by-layer (LbL) polyelectrolyte coated glass substrates
    1. Prepare 5 mg ml-1 aqueous solutions of PAH and PSS polymers (5 ml each).
    2. Drop 50 µl of PAH solution onto the central part of a dust-free glass slide, incubate for 20 min in a humidified chamber (any tightly closed glass or plastic container with moist cotton pad might be used to prevent the premature evaporation), wash several times with water.
    3. Repeat the same using PSS solution.
    4. Repeat steps A2 and A3 until the resulting coating architecture is (PAH/PSS)12PAH (12 consecutive PAH/PSS bilayers followed by a final PAH layer). The outmost polyelectrolyte should be PAH to ensure the positive charge of the deposited film. More information on fabrication of LbL films can be found elsewhere (Lvov et al., 1995).

  2. Caenorhabditis elegans nematodes cultivation
    1. Cultivate wild type C. elegans (N2 Bristol) nematodes on NGM-supplemented agar plates at 20 °C using a thermostated shaker according to a standard protocol (Brenner, 1974). Use Escherichia coli OP50 bacteria to feed worms. Observe the animals using a stereomicroscope after 48 h to detect gravid nematodes.
    2. Wash off gravid adult hermaphrodites with M9 buffer (see Recipes) into a tube and harvest by centrifugation for 2 min (1,100 x g, applicable to eggs, larvae and adult hermaphrodites). Dissolve the animals using aqueous 2% NaOCl/0.45 M NaOH solution (4.5 ml) to obtain nematode eggs. Wash the eggs with M9 buffer several times, then collect by centrifugation as described above and place onto the sterile NGM agar plates.
    3. Cultivate the nematodes at 20 °C to obtain synchronised adult hermaphrodites. Alternatively, cultivate the worms for shorter time to obtain larvae animals (Brenner, 1974).
    4. Collect the nematodes as described in step B2. Euthanise the collected worms with 45 mM aqueous levamisole hydrochloride. Wash consequently with water.
    5. Fixate the animals using 2 ml of buffered glutaraldehyde (in M9 buffer) for 2 h at 25 °C, then wash with water.

  3. Atomic force microscope setup
    1. It is assumed that the AFM operator is familiar with the AFM working principles and has previous experience with Dimension FastScan Bio AFM. For introduction into PeakForce Tapping AFM mode refer to microscope manual or to the following publication (Durkovic et al., 2014)
    2. AFM should be initiated before the sample is ready for imaging (see below). Ideally, these steps should be performed at the same time.
    3. Switch on the microscope and the operating software.
    4. Install and calibrate the mechanical properties of the probe (ScanAsyst-Fluid) according to the manufacturer’s manual. Start calibrating in air, then repeat in water.

  4. Atomic force microscopy imaging
    1. Place a droplet (100 µl) of water-suspended fixated nematodes onto the LbL-coated glass slides.
    2. Wait 30 min for settling and attaching the animals to the LbL polyelectrolyte film.
    3. Remove the excess water from the glass surface by pipetting, then wash the slide with water 3 times to remove any loosely immobilised worms.
    4. Add 200 µl of water on the top of the LbL-deposited film retaining the worms. The adhesion of the animals to the glass slide can be checked using a stereomicroscope prior to AFM imaging.
    5. Secure the glass slide on the microscope stage using vacuum suction. This should be done with due care to avoid removing the liquid from the glass slide.
    6. Locate a worm using the internal AFM optical camera. The worms will appear on the computer screen connected to the AFM internal camera as they would be seen under a stereomicroscope (Figure 1).


      Figure 1. Immobilisation of nematodes on the support film. A sketch demonstrating immobilisation of an adult C. elegans nematode on (PAH/PSS)12PAH LbL-deposited film and subsequent AFM imaging in liquid (A). Optical microscopy camera snapshot demonstrating the approach of the AFM probe to head (B), body (C) and tail (D) region of an immobilised C. elegans adult hermaphrodite.

    7. Position the probe above a certain area of the worm (i.e., head, tail or body regions).
    8. Approach the tip to the surface. Use relatively low scanning resolution (i.e., 64 lines) for locating the cuticle surface.
    9. After locating the region of interest (ROI), reduce the scanning frequency (0.8-0.9 Hz) and peak force setpoint (1 nN); increase the scanning resolution (up to 512 lines). The typical AFM images of an adult C. elegans nematode are shown in Figure 2.


      Figure 2. PeakForce Tapping AFM images of an adult C. elegans nematode inmmobilised on (PAH/PSS)12PAH LbL-deposited film: tail region (upper row) and body region (lower row). A and D. Height sensor topography images; B and E. Peak force error images; C and F. 3-D rendering of height sensor data with peak force error data overlayed.

    10. Collect images in the following channels: height sensor, peak force error, adhesion and Sneddon modulus mode. Figure 3 demonstrates the images obtained in all four channels.


      Figure 3. PeakForce Tapping AFM images of L1 larvae (A) and adult C. elegans (B) nematode imaged in water in topography (height sensor), peak force error, adhesion and modulus (Sneddon model) channels. Scale bars = 1 µm.

    11. Scans can be obtained using higher aspect ratios (i.e., 5-6), which allows increasing the longitude of the images (compare aspect ratios in Figures 2 and 3).
    12. For statistically-significant quantitative evaluation examine 5-10 individual randomly selected nematodes.

  5. Atomic force microscopy raw data processing
    1. Export raw data files to NanoScope Analysis software.
    2. Open a file, then flatten the image using 3d flattening order.
    3. Adjust colour scale using ‘Color Bar Scale Relative to Minimum Data Cursor’ function.
    4. Export high-resolution tiff files using Journal Quality Export function.
    5. For advanced image processing and nanomechanical characterisation refer to the NanoScope Analysis software operation manual.

Recipes

  1. M9 buffer
    22 mM KH2PO4
    42 mM Na2HPO4
    85.5 mM NaCl
    1 mM MgSO4

Acknowledgments

This study was performed according to the Russian Government Program of Competitive Growth of Kazan Federal University. The authors acknowledge the RFBR 17-04-02182А grant. This protocol was adapted from our previous publication (Fakhrullina et al., 2017). We thank Ms. E. Dubkova for technical help. The authors have no conflicts of interest.

References

  1. Allen, M. J., Kanteti, R., Riehm, J. J., El-Hashani, E. and Salgia, R. (2015). Whole-animal mounts of Caenorhabditis elegans for 3D imaging using atomic force microscopy. Nanomedicine 11(8): 1971-1974.
  2. Alsteens, D., Dupres, V., Yunus, S., Latge, J. P., Heinisch, J. J. and Dufrene, Y. F. (2012). High-resolution imaging of chemical and biological sites on living cells using peak force tapping atomic force microscopy. Langmuir 28(49): 16738-16744.
  3. Beaussart, A., El-Kirat-Chatel, S., Fontaine, T., Latge, J. P. and Dufrene, Y. F. (2015). Nanoscale biophysical properties of the cell surface galactosaminogalactan from the fungal pathogen Aspergillus fumigatus. Nanoscale 7(36): 14996-15004.
  4. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
  5. Durkovic, J., Kardosova, M. and Lagana, R. (2014). Imaging and measurement of nanomechanical properties within primary xyleme cell walls of broadleaves. Bio Protoc 4 (24) e1360.
  6. Fakhrullina, G., Akhatova, F., Kibardina, M., Fokin, D. and Fakhrullin, R. (2017). Nanoscale imaging and characterisation of Caenorhabditis elegans epicuticle using atomic force microscopy. Nanomedicine 13(2): 483-491.
  7. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669): 806-811.
  8. Kenyon, C. J. (2010). The genetics of ageing. Nature 464(7288): 504-512.
  9. Lvov, Y., Ariga, K., Ichinose, I. and Kunitake, T. (1995). Assembly of multicomponent protein films by means of electrostatic layer-by-layer adsorption. J Am Chem Soc 117(22): 6117-6123.
  10. O’Reilly, L. P., Luke, C. J., Perlmutter, D. H., Silverman, G. A. and Pak, S. C. (2014). C. elegans in high-throughput drug discovery. Adv Drug Deliv Rev 69-70: 247-253.
  11. Sterken, M. G., Snoek, L. B., Kammenga, J. E. and Andersen, E. C. (2015). The laboratory domestication of Caenorhabditis elegans. Trends Genet 31(5): 224-231.
  12. Stroustrup, N., Anthony, W. E., Nash, Z. M., Gowda, V., Gomez, A., Lopez-Moyado, I. F., Apfeld, J. and Fontana, W. (2016). The temporal scaling of Caenorhabditis elegans ageing. Nature 530(7588): 103-107.
  13. Swierczek, N. A., Giles, A. C., Rankin, C. H. and Kerr, R. A. (2011). High-throughput behavioral analysis in C. elegans. Nat Methods 8(7): 592-598.

简介

自由生活的土壤线虫秀丽隐杆线虫已经成为生物学中一个标志性的实验动物模型。这种透明的动物可以很容易地使用光学显微镜成像,以可视化其器官,组织,单细胞和亚细胞事件。 C的角质层。已经使用透射和扫描(SEM)电子显微镜在纳米级研究了线虫线虫。结果,由于将蠕虫嵌入树脂或用导电金层涂覆蠕虫,可能出现成像伪像。另外,固定和对比也可能损伤角质层。传统的敲击模式原子力显微镜(AFM)可用于对干燥的线虫的角质层在空气中成像,然而这种方法也存在成像缺陷。理想情况下,线虫应该在类似自然环境的条件下成像。最近,我们报道了使用PeakForce攻丝AFM模式来成功实现可视化和数值表征。线虫角质层在空气和液体中均有表达(Fakhrullina et al。,2017)。我们成像的主要线虫表面结构和角质层的力学性能表征。该协议提供了对液体浸泡的AF的AFM成像的详细描述。线虫使用PeakForce攻丝原子力显微镜线虫。
【背景】自由生活和寄生虫的线虫由于其对农业和人类福祉的显着生物学效应而受到广泛的研究。显然,线虫中研究最多和最着名的物种是自由生活的土壤线虫秀丽隐杆线虫(Sterken等人,2015年)。这种蠕虫在许多研究中已被成功地用作多功能模式生物体(Fire等人,1998; Kenyon,2010; Swierczek等人,2011; O'Reilly等人,2014; Stroustrup等人,2016年),最终确保了2002年诺贝尔奖 - 悉尼•布伦纳,约翰•苏尔斯顿和罗伯特•霍维茨。 ℃。线虫线虫是一种很小(〜1毫米长)的透明动物,可以通过光学显微镜很容易地观察到。它的角质层虽然薄而透明,是蠕虫和栖息地之间的主要保护屏障。当病原菌定居时,角质层是疾病的重要标志,导致被感染的动物死亡。此外,角质层的结构可能表明线虫的老化。从人类保健和工业角度来看,监测线虫角质层可能有助于阐明影响角质层的新型抗线虫化学物质。因此,在自然液体环境中线虫角质层的纳米级成像可能为生物医学研究开辟了新的途径。

以前,已经使用电子显微镜(透射和扫描)研究了线虫角质层(主要使用线虫作为模式生物)。不幸的是,电子显微镜不允许在液体中对蠕虫进行成像,这是它们的原生环境。用于电子显微镜的样品制备需要固定,脱水,对比染色或表面溅射,以及用于超薄切片的树脂嵌入。因此,线虫角质层可能会出现二次伪影,妨碍评估天然表面结构和机械特性。已经建立了原子力显微镜作为原位成像生物样品的有力工具,包括在液体介质中对活细胞成像(Beaussart等人,2015)。最近,应用攻丝模式AFM来可视化C。已经报道了线虫在空气中的报道(Allen等人,2015)。在空气中成像遭受与扫描电子显微镜相同的缺点,例如,图像显示典型的由脱水和空气成像造成的收缩和塌陷的角质层表面。我们设想了一种基于PeakForce攻丝AFM模式(Alsteens et al。,2012)的成功可视化的不同技术。线虫在水中的线虫角质层(Fakhrullina et al。,2017)。在这里,我们报告这个技术的详细协议。

关键字:原子力显微镜检查, 线虫, 秀丽隐杆线虫, 角质层, 峰值力轻敲, 层层组装

材料和试剂

注:除非另有说明,否则所有化学品均购自Sigma-Aldrich。

  1. 移液器提示
  2. 培养皿
  3. 无尘Nexterion玻璃幻灯片(肖特)
  4. 野生型线虫(N2布里斯托尔)线虫
  5. 大肠杆菌 OP50细菌
  6. 聚(烯丙胺盐酸盐)(PAH,分子量〜17.5kDa)(Sigma-Aldrich,目录号:283215)
  7. 聚(4-苯乙烯磺酸钠)(PSS,分子量约70kDa)(Sigma-Aldrich,目录号:243051)
  8. 线虫生长培养基(NGM)(bioWORLD,目录号:30620040-1)
  9. 线虫裂解液(2%NaOCl水溶液/0.45M NaOH)
  10. 次氯酸钠(NaClO)(Sigma-Aldrich,目录号:425044)
  11. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  12. 盐酸左旋咪唑(Sigma-Aldrich,目录号:L9756)
  13. 戊二醛(25%水溶液)(Sigma-Aldrich,目录号:G5882)
  14. 通过Simplicity(Millipore)水净化系统纯化的超纯水(类型1)
  15. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:NIST200B)
  16. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:71649)
  17. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  18. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:793612)
  19. M9缓冲水溶液(见食谱)

设备

  1. 移液器(机械Eppendorf或吉尔森移液器)
  2. Eppendorf Scientific Excella E24温控台式摇床(Eppendorf,New Brunswick TM,型号:Excella E24)
  3. Biosan V-1 plus涡旋(Biosan,型号:V-1 plus)
  4. 尼康SMZ 745 T立体显微镜(尼康,型号:SMZ745T)
  5. 尺寸FastScan生物原子力显微镜(布鲁克,型号:Dimension FastScan)配备液体潜入式扫描仪
    注意:不要使用空气扫描仪扫描液体,这可能会损坏仪器。
  6. Biosan Microspin 12离心机(Biosan,型号:Microspin 12)
  7. ScanAsyst-Fluid探针(标称长度70μm,尖端半径20nm,弹簧常数0.7N m -1)(Bruker,型号:SCANASYST-FLUID) 注意:也可以使用Bruker或其他生产商提供的其他AFM探头,这些探头经批准可以在PeakForce Tapping模式下使用,其特性类似于ScanAsyst-Fluid探头。

软件

  1. NanoScope原子力显微镜操作软件(布鲁克公司)
  2. NanoScope分析v.1.7。软件(布鲁克公司)

程序

  1. 制备逐层(LbL)聚电解质涂覆的玻璃基材
    1. 准备5毫克ml-1 PAH和PSS聚合物水溶液(每个5毫升)。
    2. 将50μlPAH溶液滴在无尘载玻片的中心部位,在加湿室中孵育20 min(任何密封的玻璃或带有湿棉垫的塑料容器都可能用于防止过早蒸发)次与水。

    3. 使用PSS解决方案重复相同的操作
    4. 重复步骤A2和A3,直到得到的涂层结构为(PAH / PSS)12 PAH(连续12个PAH / PSS双层,然后是最终的PAH层)。最外面的聚电解质应该是PAH以确保沉积膜的正电荷。有关制造LbL薄膜的更多信息可以在别处找到(Lvov等人,1995)。

  2. 秀丽隐杆线虫线虫种植
    1. 培养野生型C.根据标准方案(Brenner,1974),使用恒温振荡器,在20℃下,在NGM补充的琼脂平板上的线虫(N2布里斯托尔)线虫。使用大肠杆菌 OP50细菌喂食蠕虫。
      使用立体显微镜观察动物48小时后检测妊娠线虫
    2. 用M9缓冲液(见食谱)将成年妊娠的成熟雌雄同体洗净并放入离心管中,离心2分钟(1100微克/克,适用于卵,幼虫和成年雌雄同体)收获。使用2%NaOCl /0.45M NaOH水溶液(4.5ml)溶解动物以获得线虫卵。用M9缓冲液洗蛋几次,然后如上所述通过离心收集并置于无菌NGM琼脂平板上。
    3. 在20℃培养线虫以获得同步的成年雌雄同体。或者,培养蠕虫的时间较短,以获得幼虫动物(Brenner,1974)。
    4. 收集步骤B2中所述的线虫。安乐死收集的蠕虫与45毫米左旋咪唑盐酸盐水溶液。用水洗净。
    5. 使用2毫升缓冲戊二醛(在M9缓冲液中)在25°C固定动物2小时,然后用水洗。

  3. 原子力显微镜设置
    1. 假定AFM操作员熟悉AFM的工作原理,并且具有Dimension FastScan Bio AFM的先前经验。有关引入PeakForce攻丝AFM模式的信息,请参阅显微镜手册或以下出版物(Durkovic ,2014)
    2. 在样品准备好成像之前应该启动AFM(见下文)。理想情况下,这些步骤应该同时进行。
    3. 打开显微镜和操作软件。
    4. 根据制造商的手册安装并校准探头(ScanAsyst-Fluid)的机械特性。开始在空气中校准,然后在水中重复。

  4. 原子力显微镜成像
    1. 放置液滴(100微升)的水悬浮固定线虫到LbL涂层载玻片上。
    2. 等待30分钟沉降并将动物附着在LbL聚电解质膜上。
    3. 通过移液将玻璃表面多余的水分移除,然后用水清洗玻片3次,以除去任何松散固定的蠕虫。
    4. 在保留蠕虫的LbL沉积膜的顶部添加200μl水。
      在AFM成像之前,使用立体显微镜可以检查动物与载玻片的粘附
    5. 使用真空抽吸将载玻片固定在显微镜台上。这应该小心,以避免从载玻片上的液体。
    6. 使用内部AFM光学相机找到蠕虫。蠕虫会出现在连接到AFM内部摄像头的电脑屏幕上,就像在立体显微镜下看到的那样(图1)。


      图1.在支撑膜上固定线虫。 显示成年人C的固定的草图。 (PAH / PSS)12 PAH LbL沉积膜上的线虫(elegans)线虫和随后在液体(A)中的AFM成像。光学显微镜照相机快照显示AFM探针接近固定化C的头部(B),身体(C)和尾部(D)区域。线虫成年雌雄同体

    7. 将探头放置在蠕虫的某个区域(即,头部,尾部或身体区域)。
    8. 接近尖端的表面。使用相对较低的扫描分辨率(即,64行)来定位角质层表面。
    9. 定位感兴趣区域(ROI)后,降低扫描频率(0.8-0.9 Hz)和峰值力设定值(1 nN);增加扫描分辨率(最多512行)。典型的成人AFM图像。线虫线虫如图2所示。


      图2. PeakForce拍摄成人的AFM图像(PAH / PSS)12 PAH LbL沉积膜:尾部区域(上部行)和体部区域(下部行)上的线虫线虫。 A和D高度传感器地形图像; B和E.峰值误差图像; C和F.高度传感器数据的三维渲染,叠加峰值误差数据。

    10. 在以下通道中采集图像:高度传感器,峰值力误差,附着力和Sneddon模量模式。图3演示了在所有四个通道中获得的图像。


      图3. PeakForce攻击L1幼虫(A)和成虫C的AFM图像。线虫(B)在地形(高度传感器),峰值力误差,粘附力和模量(Sneddon模型)通道中在水中成像的线虫。比例尺=1μm。

    11. 可以使用更高的纵横比(即,5-6)来获得扫描,这允许增加图像的经度(比较图2和图3中的纵横比)。
    12. 对于统计学显着的定量评估,检查5-10个人随机选择的线虫。

  5. 原子力显微镜原始数据处理
    1. 将原始数据文件导出到NanoScope分析软件。
    2. 打开一个文件,然后使用3 d 拼合顺序来平整图像。

    3. 使用“相对于最小数据光标的色条刻度”功能调整色标
    4. 使用Journal Quality Export功能导出高分辨率的tiff文件。
    5. 对于高级图像处理和纳米机械表征,请参考NanoScope分析软件操作手册。

食谱

  1. M9缓冲区
    22 mM KH 2 PO 4 4 42mM Na 2 HPO 4 4 85.5 mM NaCl
    1mM MgSO 4

致谢

这项研究是根据喀山联邦大学俄罗斯政府竞争力增长计划进行的。作者承认RFBR 17-04-02182授予。该协议是根据我们以前的出版物(Fakhrullina et。,2017)改编的。我们感谢E. Dubkova女士的技术帮助。作者没有利益冲突。

参考

  1. Allen,M.J.,Kanteti,R.,Riehm,J.J。,El-Hashani,E。和Salgia,R。(2015)。 使用原子力显微镜进行三维成像的整个动物坐骑 Caenorhabditis elegans 。 Nanomedicine 11(8):1971-1974。
  2. Alsteens,D.,Dupres,V.,Yunus,S.,Latge,J.P.,Heinisch,J.J。和Dufrene,Y.F。(2012)。 利用峰值取力原子力显微镜对活细胞进行化学和生物学部位的高分辨成像。 / a> Langmuir 28(49):16738-16744。
  3. Beaussart,A.,El-Kirat-Chatel,S.,Fontaine,T.,Latge,J.P。和Dufrene,Y.F。(2015)。 来自真菌病原体细胞表面的半乳糖氨基半乳聚糖的纳米级生物物理性质烟曲霉 。 Nanoscale 7(36):14996-15004。
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  6. Fakhrullina,G.,Akhatova,F.,Kibardina,M.,Fokin,D。和Fakhrullin,R.(2017)。 使用原子力显微镜对秀丽隐杆线虫上皮细胞进行纳米级成像和表征。 / a> Nanomedicine 13(2):483-491。
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  9. Lvov,Y.,Ariga,K.,Ichinose,I。和Kunitake,T。(1995)。 通过静电逐层吸附装配多组分蛋白质膜 < J Am Chem Soc 117(22):6117-6123。
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  12. Stroustrup,N.,Anthony,W.E.,Nash,Z.M。,Gowda,V.,Gomez,A.,Lopez-Moyado,I.F.,Apfeld,J.and Fontana,W。(2016)。 秀丽隐杆线虫(Caenorhabditis elegans)的时间缩放老化。 自然 530(7588):103-107。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Akhatova, F., Fakhrullina, G., Gayazova, E. and Fakhrullin, R. (2017). Nematode Epicuticle Visualisation by PeakForce Tapping Atomic Force Microscopy. Bio-protocol 7(21): e2596. DOI: 10.21769/BioProtoc.2596.
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