三维细胞外囊泡的直接随机光学重建显微镜
Summary
直接随机光学重建显微镜（dSTORM）用于绕过光学显微镜的典型衍射极限，并在纳米尺度上观察外泌体。它可以在二维和三维空间中用于表征外泌体。
Abstract
细胞外囊泡（EV）由所有细胞类型释放，并在细胞信号传导和体内平衡中起重要作用。电动汽车的可视化通常需要间接方法，因为它们的直径很小（40-250nm），低于典型光学显微镜的衍射极限。我们开发了一种基于超分辨率显微镜的电动汽车可视化，以绕过二维和三维的衍射极限。使用这种方法，我们可以将EV的三维形状解析为XY轴上的+/- 20nm分辨率和沿Z轴的+/- 50nm分辨率。总之，我们建议将超分辨率显微镜视为EV的表征方法，包括外泌体以及包膜病毒。
Introduction
细胞外囊泡（EV）是由所有细胞类型释放的膜结合囊泡。它们含有脂质，蛋白质，代谢物和核酸，并在细胞之间局部转移这些物质，并在组织和器官之间远端转移。EV有三种主要亚型：凋亡体，微囊泡和外泌体1，2。在这里，我们将讨论重点放在外泌体及其相关蛋白质上。
外泌体是分泌的囊泡，起源于早期内体向内萌芽进入多泡体（MVB）。然后，MVB与质膜融合，将外泌体释放到细胞外空间以传播到其他细胞3，4。外泌体存在于40至150nm的范围内，并富含称为四泛素（CD9，CD63，CD81）的内体跨膜蛋白，转运所需的膜结合内体分选复合物（ESCRT）和脂筏相关蛋白1，2，5，6，7。
表征外泌体的生化组成已成为研究人员更好地了解其功能性质的热门领域。存在许多用于可视化和表征外泌体的方法，包括纳米级流式细胞术，纳米颗粒跟踪分析（NTA），扫描和透射电子显微镜（TEM），表面等离子体共振，电阻脉冲传感和传统光学显微镜，每种方法都包含固有的优缺点8，9。TEM和冷冻电镜可以达到基于纳米的分辨率，但往往需要脱水和冷冻断裂步骤，从而缩小或裂解EV10、11。NTA依赖于光散射，允许一次表征数百个EV，但是对颗粒尺寸的间接测量，并且不能轻易区分EV，病毒和蛋白质聚集体12，13，14，15，16。纳米级流式细胞术采用来自激发路径的光散射，然后可以将其转换为尺寸测量，但这是一种新兴技术，并且对于各种仪器12，17，18的线性范围内的颗粒大小几乎没有共识。
使用荧光蛋白或染料的传统光学显微镜一直是可视化细胞内亚细胞区室，蛋白质复合物和信号传导机制的最常用技术之一。虽然这种技术被证明在可视化复合物的定位方面是有用的，但传统光学显微镜的衍射极限（约250-400nm）阻止了蛋白质或结构在外泌体（40-150nm）的典型尺寸范围内的清晰分辨率12，19，20。
超分辨率显微镜，即直接随机光学重建显微镜（dSTORM），通过利用特定荧光团的光开关特性并检测这些闪烁事件来重建精度低至纳米精度的图像，从而将自己与传统的光学显微镜区分开来21。在数以万计的单个曝光过程中，使用高帧率检测相机收集光开关事件，并使用点扩散函数以高置信度绘制光开关荧光团19，20，22的确切位置。这使得dSTORM绕过了光学显微镜的衍射极限。几个小组已经报道了使用超分辨率技术来可视化和跟踪外泌体及其相关蛋白质22，23，24，25。最终分辨率取决于荧光团的生物物理性质，但通常沿XY轴的范围为+/-10-100nm，允许单分子分辨率。
在XY轴上以这种尺度分辨单个荧光团的能力已经彻底改变了显微镜。然而，关于外泌体的三维（3-D）dSTORM的数据很少。因此，我们试图建立一种标准操作程序（SOP），用于基于dSTORM的纯化EV的可视化和表征，包括外泌体到3D的纳米精度。
Protocol
1 细胞系的繁殖和维护 获取人骨肉瘤细胞（U2OS），并将细胞置于补充有10%外泌体游离胎牛血清和1x青霉素/链霉素溶液的生长培养基中。注意：无外泌体的胎牛血清是按照McNamara等人提出的方案产生的。26. 将U2OS细胞在37°C的铜包衣培养箱中保持在5%CO2中，并在T175烧瓶26，27中传代细胞。细胞必须保持在对数生长?…
Representative Results
本研究的目的是评估超分辨率显微镜在三维（3-D）中以纳米分辨率可视化单个电动汽车方面的有效性。为了分析单个EV的形状和大小，我们采用了光开关染料，并用远红色的膜插层染料孵育EV，并通过色谱法29除去多余的染料。然后在640 nm激发激光下在超分辨率显微镜下观察亲和捕获的抗CD81和红色染色的EV。在校准显微镜后，XY轴上产生16 nm的平均误差，Z轴上产生38 nm的平均误差（…
Discussion
EV已成为一个受欢迎的研究领域，因为它们在许多细胞内过程和细胞间信号传导中起着重要作用1，30。然而，它们的可视化被证明是困难的，因为它们的小尺寸低于光学显微镜的衍射极限。直接随机光学重建显微镜（dSTORM）是一种直接的可视化方法，它通过捕获单个荧光团随时间推移的光开关事件并根据这些闪烁事件重建图像来绕过衍射极限<sup class="xre…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢Oxford Nanoimaging的建设性反馈和指导。这项工作由5UM1CA121947-10资助给R.P.M.和1R01DA040394给D.P.D.
Materials
| 15 µ-Slide 8 well plates | Ibidi | 80827 | |
| 1X PBS | Gibco | 14190-144 | |
| 1X Penicillin Streptomycin solution | Gibco | 15140-122 | |
| 50 mL conical tube | Thermo Fisher | 339652 | |
| 500 mL 0.22 µm vacuum filtration apparatus | Genesee | 25-227 | |
| 750 kDa hollow-fiber cartridge cutoff filter | Cytiva | 29-0142-95 | |
| AKTA Flux S | Cytiva | 29-0384-37 | |
| AKTA Start | Cytiva | 29022094-ECOMINSSW | |
| Anti-CD81 magnetic beads | Thermo Fisher | 10616D | |
| B-cubed buffer | ONI | BCA0017 | |
| CellMask Red | Thermo Fisher | C10046 | |
| Dubelco's Modified Eagle Medium | Thermo Fisher | 10566016 | |
| Fetal Bovine Serum | VWR | 97068-085 | |
| Frac 30 Fraction collector | Cytiva | 29022094-ECOMINSSW | |
| Glycine pH=2.0 | Thermo Fisher | BP381-5 | |
| HiTrap CaptoCore 700 Column | Cytiva | 17548151 | |
| Molecular Biology Grade Water | Corning | 9820003 | |
| Nanoimager | Oxford Nanoimaging | Custom | |
| Paraformaldehhyde | Electron Microscopy Sciences | 15710 | |
| Polyethylene glycol | Thermo Fisher | BP233-1 | |
| RNase A | Promega | A797C | |
| T175 Flasks | Genesee | 25-211 | |
| Tetraspek microspheres | Invitrogen | T7279 | |
| Tris- HCl pH=7.5 | Thermo Fisher | BP153-1 | |
| Unicorn V | Cytiva | 29022094-ECOMINSSW |
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Citazione di questo articolo
Chambers, M. G., McNamara, R. P., Dittmer, D. P. Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions. J. Vis. Exp. (174), e62845, doi:10.3791/62845 (2021).