原子力显微镜对细胞外囊泡的成像
Summary
描述了一个分步程序，用于从液体样品中分离体和细胞外囊泡的无标签固定，并通过原子力显微镜（AFM）进行成像。AFM 图像用于估计溶液中囊泡的大小，并描述其他生物物理特性。
Abstract
外体和其他细胞外囊泡 （EV） 是分子复合物，由脂质膜囊泡、其表面装饰（由膜蛋白和其他分子）和从母细胞（包括RNA）继承的多种发光含量组成，蛋白质和DNA。EV的流体动力学尺寸的表征，取决于由表面装饰形成的囊泡和其冠状层的大小，已成为常规。对于最小EV的外生体，水动力和囊泡大小之间的相对差异显著。低温透射电子显微镜（低温-TEM）成像（低温-TEM）成像对囊泡尺寸的表征仍然是一项挑战，因为仪器的成本、执行样品制备、成像和样品制备所需的专业知识数据分析，以及图像中经常观察到的少量粒子。原子力显微镜（AFM）是一个广泛提供且易于获取的替代方法，它可以生成关于细胞外囊泡的三维几何形状、尺寸和其他生物物理特性的通用数据。开发的协议指导用户使用此分析工具，并概述了 AFM 分析 EV 的工作流程，其中包括水合或干燥形式的成像 EV 的样品制备、静电固定基板上的囊泡、数据采集、分析和解释。具有代表性的结果表明，EV在修改后的云母表面的固定是可预测的、可定制的，并允许用户获得大量囊泡的尺寸调整结果。根据AFM数据进行囊泡尺寸的尺寸与低温-TEM成像一致。
Introduction
细胞外囊泡 （EV） 存在于所有体液中，包括血液、尿液、唾液、牛奶和羊水。外体体形成一个区域类别的EV与其他EV不同，通过内生，内生子途径的标记，和所有EV中最小的尺寸。外体体的大小经常被报告，在研究之间有很大的变异性。大小调整结果与方法相关，反映了不同分析技术用于估计EV尺寸1，2的物理原理的差异。例如，纳米粒子跟踪分析 （NTA） 是使用最广泛的尺寸表征技术，它估计了 EV 的大小作为流体动力学直径，该直径描述了溶液中 EV 对布朗流动性的阻力。囊泡的流体动力学直径越大，意味着它在液体中的流动性较低。囊泡周围的冠状层，由表面蛋白质和其他固定在膜表面或吸附的分子组成，严重阻碍流动性，增加EV的流体动力学尺寸。相对而言，这种增加对于外体体3来说特别大，如图1所示。
低温透射电子显微镜（低温-TEM）成像是描述囊泡大小和形态在其水合状态的明确技术。然而，仪器的高成本和使用它所需的专业知识正确地激发了对可成像水合电动汽车的替代技术的探索。在采集的低温-TEM 图像中观察到或特征的 EV 数量相对较少，是该技术的另一个显著缺点。
原子力显微镜（AFM）通过扫描基板上的探针来栅格显示表面粒子的图像，从而可视化水合或干燥EV4、5、6的三维地形。本研究概述了协议对AFM的EV特征的基本步骤。在液体中成像囊泡之前，必须将其固定在基板上，通过系绳到功能化表面、困在过滤器中或静电吸引力7。在带正电基板上的静电固定是一种特别方便的选择，用于对已知具有负Zeta电位的外体体进行固定。然而，固定表面细胞外囊泡的同样的静电力也会扭曲其形状，这使得成像后数据分析变得至关重要。我们通过描述基于固定在表面上的外体体变形形状的AFM数据估计溶液中球状囊泡大小的算法来阐述这一点。
在已开发的协议中，介绍了囊泡强稳定处理程序，并随后介绍了在水合或干燥状态下执行原子力成像所需的步骤。确定了影响固定囊泡表面浓度的因素。指导如何对溶液中不同浓度的EV样品进行静电固定。讨论了基于足够数量的固定囊泡估计不同生物物理特性的经验概率分布的实验条件的选择。给出了AFM数据成像后分析的例子。具体来说，描述了一种算法，用于根据固定性EV的AFM特征来确定溶液中囊泡的大小。具有代表性的结果表明AFM的囊泡大小与低温-TEM成像结果的一致性。
Protocol
Representative Results
Discussion
从生物流体、表面扫描和图像分析中固定 EV 是开发中 EV 在液体中的 AFM 表征协议的基本步骤。与图像表面积和固定在基板上的囊泡的表面浓度一起，可适应AFM成像的囊泡数量。鉴于EV和外兆体18的负zeta电位，我们提倡将EV从液体样品静态固定到AFM基板。当表面充满正电荷时，固定是有效的。在EV固定之前，可能需要向基材提供正表面电荷，如云母-一般公式KAl 2（AlSi3O10…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢国家科学基金会（奖励号IGERT-0903715）、犹他大学（化学工程系种子赠款和研究生研究奖学金）和斯科尔科沃科学研究所的财政支持。与技术（科技奖学金）。
Materials
| AFM/STM Controller | Bruker | Multimode Nanoscope V | This AFM controller supports imaging of biological samples in liquid and air. |
| AFM/STM metal specimen discs (10 mm) | TedPella | 16207 | Metal specimen disc on which a mica disk is attached by a double-sided tape or other means. |
| AFM/STM Mica discs (10 mm) | TedPella | 50 | Highest quality grade V1 mica, 0.21mm (0.0085”) thick. Interleaved, in packages of 10. Can be mounted on AFM/STM discs. Available in four diameters |
| AFM probe for imaging in the air | Bruker | TESP-V2 | High quality etched silicon probes for tapping mode and non-contact mode for scanning in the air. |
| AFM probe for soft sample imaging in liquid | Bruker | MLCT | Soft silicon nitride cantilevers with silicon nitride tips, which are well-suited for liquid operation. The range in force constants enables users to image extremely soft samples in contact mode as well as high load vs distance spectroscopy. |
| Double sided tape | Spectrum | 360-77705 | Used to fix the mica disk on the metal specimen disc. |
| ExoQuick-TC | System Biosciences | EXOTC50A-1 | ExoQuick-TC is a proprietary polymer-based kit designed for exosome isolation from tissue culture media. |
| Glass probe AFM holder for imaging in liquid | Bruker | MTFML-V2 | This glass probe holder is designed for scanning in fluid with the MultiMode AFM. The holder can be used in peak force tapping mode, contact mode, tapping mode, and force modulation. The probe is acoustically driven by a separate piezo oscillator for larger amplitude modulation. The holder is supplied with two ports, required fittings, and accessories kit for adding and removing fluids. |
| Gwyddion | Czech Metrology Institute. | Version 2.52 | Open Source software for visualization and analysis of data fields obtained by scanning probe microscopy techniques. |
| Lint-free blotting paper | GE Healthcare Whatman | Grade GB003 Blotting Paper | Use this blotting paper to remove NiCl2 after the modification of the mica's substrate. |
| Lint-free cleanroom wipes | Texwipe | AlphaWipe TX1004 | Use these polyester wipes for surface cleaning. |
| Nickel(II) chloride (NiCl2) | Sigma-Aldrich | 339350 | Powder used to make 10 mM NiCl2 in DI water |
| Phosphate Buffered Saline (1x) | Gibco | 10010023 | PBS, pH 7.4 |
References
- Chernyshev, V. S., et al. Size and shape characterization of hydrated and desiccated exosomes. Analytical and Bioanalytical Chemistry. 407, 3285-3301 (2015).
- Ramirez, M. I., et al. Technical challenges of working with extracellular vesicles. Nanoscale. 10, 881-906 (2018).
- Skliar, M., et al. Membrane proteins significantly restrict exosome mobility. Biochemical and Biophysical Research Communications. 501, 1055-1059 (2018).
- Parisse, P., et al. Atomic force microscopy analysis of extracellular vesicles. European Biophysics Journal. 46, 813-820 (2017).
- Ito, K., et al. Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning. Journal of Physical Chemistry B. 122, 6224-6235 (2018).
- Sharma, S., LeClaire, M., Gimzewski, J. K. Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles. Nanotechnology. 29, 132001 (2018).
- Meyer, R. L. Immobilisation of living bacteria for AFM imaging under physiological conditions. Ultramicroscopy. 110, 1349-1357 (2010).
- Théry, C., Amigorena, S., Raposo, G., Clayton, A., et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current protocols in cell biology. Chapter 3 (Unit 3.22), (2006).
- Taylor, D. D., Zacharias, W., Gercel-Taylor, C. Exosome isolation for proteomic analyses and RNA profiling. Methods in Molecular Biology. 728, 235-246 (2011).
- Théry, C., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 8, 1535750 (2019).
- Weng, Y., et al. Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling. The Analyst. 141, 4640-4646 (2016).
- Nečas, D., Klapetek, P. Gwyddion: an open-source software for SPM data analysis. Open Physics. 10, 181-188 (2012).
- Hsueh, C., Chen, H., Gimzewski, J. K., Reed, J., Abdel-Fattah, T. M. Localized nanoscopic surface measurements of nickel-modified Mica for single-molecule DNA sequence sampling. ACS Applied Materials and Interfaces. 2, 3249-3256 (2010).
- Conde-Vancells, J., et al. Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. Journal of Proteome Research. 7, 5157-5166 (2008).
- Zhou, Y., et al. Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem Cell Research & Therapy. 4, 34 (2013).
- Coleman, B. M., Hanssen, E., Lawson, V. A., Hill, A. F. Prion-infected cells regulate the release of exosomes with distinct ultrastructural features. FASEB Journal. 26, 4160-4173 (2012).
- Briegel, A., et al. Universal architecture of bacterial chemoreceptor arrays. Proceedings of the National Academy of Sciences. 106, 17181-17186 (2009).
- Akagi, T., et al. On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells. PLOS ONE. 10, e0123603 (2015).
- Sharma, S., et al. Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano. 4, 1921-1926 (2010).
- Radeghieri, A., et al. Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles. Biochemical and Biophysical Research Communications. 483, 706-711 (2017).
- Woo, J., Sharma, S., Gimzewski, J. The Role of Isolation Methods on a Nanoscale Surface Structure and Its Effect on the Size of Exosomes. Journal of Circulating Biomarkers. 5, 11 (2016).
- Deegan, R. D., et al. Capillary flow as the cause of ring stains from dried liquid drops. Nature. 389, 827-829 (1997).
- Johnson, C. A., Lenhoff, A. M. Adsorption of Charged Latex Particles on Mica Studied by Atomic Force Microscopy. Journal of Colloid and Interface Science. 179, 587-599 (1996).
- Pastré, D., et al. Adsorption of DNA to Mica Mediated by Divalent Counterions: A Theoretical and Experimental Study. Biophysical Journal. 85, 2507-2518 (2003).
- van der Pol, E., Böing, A. N., Harrison, P., Sturk, A., Nieuwland, R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacological Reviews. 64, 676-705 (2012).
