Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3 Women's Centre, John Radcliffe Hospital, Oxford, UK;
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3 Women's Centre, John Radcliffe Hospital, Oxford, UK.
J Extracell Vesicles. 2014 Nov 24;3:25361. doi: 10.3402/jev.v3.25361. eCollection 2014.
Optical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs.
NTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values.
The following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364-1.375 and 1.398-1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV.
Using NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37-1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles.
This method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.
光学技术常用于测量细胞外囊泡（EV）的大小和数量。为了比较不同方法和实验室的数据，合适的校准标准至关重要。合适的校准标准的折射率（RI）必须尽可能接近 EV，但 EV 的 RI 目前尚不清楚。要测量 EV 的 RI，需要准确了解大小和光散射。由于大多数 EV 无法通过光学显微镜分辨，并且其直径小于可见光的波长，因此很难测量这两个参数。然而，纳米颗粒跟踪分析（NTA）可以提供大小和相对光散射强度（rLSI）值。因此，我们试图确定是否可以使用 NTA 测量单个 EV 的 RI。
使用 NTA 测量具有已知大小和 RI（分别为 1.470 和 1.633）的聚苯乙烯和二氧化硅微球以及从多种细胞中分离的 EV 的 rLSI 和大小。我们开发了一种基于 Mie 散射代码的软件，可根据 rLSI 数据计算颗粒 RI。该模型为具有已知大小（100nm 和 200nm）和 RI 的聚苯乙烯和二氧化硅微球的理论散射强度建模。使用聚苯乙烯和二氧化硅微球的数据验证了该模型。该软件对每个囊泡的大小和 rLSI 数据进行处理，以生成 RI 值。
获得以下模态 RI 测量值：新鲜尿液 EV 为 1.374，冻干尿液 EV 为 1.367，神经母细胞瘤 EV 为 1.393，血液 EV 为 1.398，激活血小板 EV 为 1.390，小胎盘 EV 为 1.364-1.375 和 1.398-1.414（>200nm）用于大胎盘 EV。与小胎盘 EV 相比，大胎盘 EV 的 RI 明显更高（p<0.0001）。小 EV 的 RI 值分布范围比更异质的大 EV 更窄。
使用 NTA 和 Mie 散射理论，我们证明了使用 NTA 数据估算亚微米 EV 的 RI 是可行的。EV 的模态 RI 通常为 1.37-1.39，而对于一些大于 200nm 的大（>200nm）微囊泡，观察到 RI 值大于 1.40。
这种测量 EV RI 的方法将有助于为 EV 测量开发合适的校准标准。
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