Extracellular vesicles (EVs) are commonly studied by flow cytometry. Due to their small size and low refractive index, the scatter intensity of most EVs is below the detection limit of common flow cytometers. Here, we aim to improve forward scatter (FSC) and side scatter (SSC) sensitivity of a common flow cytometer to detect single 100 nm EVs. The effects of the optical and fluidics configuration on scatter sensitivity of a FACSCanto (Becton Dickinson) were evaluated by the separation index (SI) and robust coefficient of variation (rCV) of polystyrene beads (BioCytex). Improvement is defined as increased SI and/or reduced rCV. Changing the obscuration bar improved the rCV 1.9‐fold for FSC. A 10‐fold increase in laser power improved the SI 19‐fold for FSC and 4.4‐fold for SSC, whereas the rCV worsened 0.8‐fold and improved 1.5‐fold, respectively. Confocalization worsened the SI 1.2‐fold for FSC, and improved the SI 5.1‐fold for SSC, while the rCV improved 1.1‐fold and worsened 1.5‐fold, respectively. Replacing the FSC photodiode with a photomultiplier tube improved the SI 66‐fold and rCV 4.2‐fold. A 2‐fold reduction in sample stream width improved both SI and rCV for FSC by 1.8‐fold, and for SSC by 1.3‐ and 2.2‐fold, respectively. Decreasing the sample flow velocity worsened rCVs. Decreasing the flow channel dimensions and the pore size of the sheath filter did not substantially change the SI or rCV. Using the optimal optical configuration and fluidics settings, the SI improved 3.8∙10⁴‐fold on FSC and 30‐fold on SSC, resulting in estimated detection limits for EVs (assuming a refractive index of 1.40) of 246 and 91 nm on FSC and SSC, respectively. Although a 50‐fold improvement on FSC is still necessary, these adaptions have produced an operator‐friendly, high‐throughput flow cytometer with a high sensitivity on both SSC and FSC. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

中文翻译：

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一种提高流式细胞仪检测细胞外囊泡散射灵敏度的系统方法。

细胞外囊泡 (EV) 通常通过流式细胞术进行研究。由于体积小、折射率低，大多数 EV 的散射强度低于普通流式细胞仪的检测限。在这里，我们旨在提高普通流式细胞仪的前向散射 (FSC) 和侧向散射 (SSC) 灵敏度，以检测单个 100 nm EV。通过聚苯乙烯珠粒 (BioCytex) 的分离指数 (SI) 和稳健变异系数 (rCV) 评估光学和流体配置对 FACSCanto (Becton Dickinson) 散射灵敏度的影响。改善定义为 SI 增加和/或 rCV 减少。更改遮蔽条将 FSC 的 rCV 提高了 1.9 倍。激光功率增加 10 倍，FSC 的 SI 提高了 19 倍，SSC 的 SI 提高了 4.4 倍，而 rCV 分别恶化了 0.8 倍和提高了 1.5 倍。共聚焦使 FSC 的 SI 恶化了 1.2 倍，SSC 的 SI 提高了 5.1 倍，而 rCV 分别提高了 1.1 倍和恶化了 1.5 倍。用光电倍增管代替 FSC 光电二极管提高了 SI 66 倍和 rCV 4.2 倍。样品流宽度减少 2 倍，FSC 的 SI 和 rCV 提高了 1.8 倍，SSC 的 SI 和 rCV 分别提高了 1.3 和 2.2 倍。降低样品流速会恶化 rCV。减小流道尺寸和鞘膜过滤器的孔径不会显着改变 SI 或 rCV。使用最佳光学配置和流体学设置，SI 提高了 3.8∙10 用光电倍增管代替 FSC 光电二极管提高了 SI 66 倍和 rCV 4.2 倍。样品流宽度减少 2 倍，FSC 的 SI 和 rCV 提高了 1.8 倍，SSC 的 SI 和 rCV 分别提高了 1.3 和 2.2 倍。降低样品流速会恶化 rCV。减小流道尺寸和鞘膜过滤器的孔径不会显着改变 SI 或 rCV。使用最佳光学配置和流体学设置，SI 提高了 3.8∙10 用光电倍增管代替 FSC 光电二极管提高了 SI 66 倍和 rCV 4.2 倍。样品流宽度减少 2 倍，FSC 的 SI 和 rCV 提高了 1.8 倍，SSC 的 SI 和 rCV 分别提高了 1.3 和 2.2 倍。降低样品流速会恶化 rCV。减小流道尺寸和鞘膜过滤器的孔径不会显着改变 SI 或 rCV。使用最佳光学配置和流体学设置，SI 提高了 3.8∙10FSC 的⁴倍和 SSC 的 30 倍，导致 EV（假设折射率为 1.40）在 FSC 和 SSC 上的估计检测限分别为 246 和 91 nm。尽管仍需要对 FSC 进行 50 倍的改进，但这些改进已经产生了对 SSC 和 FSC 均具有高灵敏度的操作友好型、高通量流式细胞仪。© 2020 作者。Cytometry Part A由 Wiley Periodicals, Inc. 代表 International Society for Advancement of Cytometry 出版。