Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy,Langmuir

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Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy
Langmuir ( IF 3.7 ) Pub Date : 2018-06-20 00:00:00 , DOI: 10.1021/acs.langmuir.7b04214
Déborah L. M. Rupert ₁ , Mokhtar Mapar ₁ , Ganesh Vilas Shelke ₂ , Karin Norling ₁ , Mathias Elmeskog ₁ , Jan O. Lötvall ₂ , Stephan Block ₁ , Marta Bally _{1,

3} , Björn Agnarsson ₁ , Fredrik Höök ₁

Affiliation

Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index (n ≈ 1.35) compared with the LD EVs (n ≈ 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.

中文翻译：

光波导散射和荧光显微镜显示的细胞外囊泡的有效折射率和脂质含量

细胞外囊泡（EVs）引起了越来越多的兴趣，这是因为它们在各种生物过程中起着关键作用，并且由于其在临床诊断中作为生物标记物以及在药物递送和基因治疗应用中作为有效载体的潜在用途而受到关注。然而，对它们的充分利用在很大程度上取决于将它们分类为不同亚群的可能性，而这一任务又依赖于有效手段来识别其独特的生物分子和物理特征。由于EV样本的异质性很大，因此此类信息仍然难以捉摸，因此需要新的和互补的表征方案，以帮助扩展独特的EV功能库。在这项工作中，我们使用具有单一EV分辨率的表面敏感波导散射显微镜来表征大小相似的EV的两个子集，这些子集是根据浮力密度的差异而预先分离的。出乎意料的是，散射强度分布显示高密度（HD）人群的散射强度平均比低密度（LD）人群的散射强度低三倍。通过用自插入脂质膜染料进一步标记EV样品，可以同时测量来自EV的散射强度和荧光强度，并在单颗粒水平上建立关联。标记的HD样品不仅显示出较低的荧光和散射强度，还显示出较低的有效折射率（出乎意料的是，散射强度分布显示高密度（HD）人群的散射强度平均比低密度（LD）人群的散射强度低三倍。通过用自插入脂质膜染料进一步标记EV样品，可以同时测量来自EV的散射强度和荧光强度，并在单颗粒水平上建立关联。标记的HD样品不仅显示出较低的荧光和散射强度，还显示出较低的有效折射率（出乎意料的是，散射强度分布表明，高密度（HD）人群的散射强度平均比低密度（LD）人群的散射强度低三倍。通过用自插入脂质膜染料进一步标记EV样品，可以同时测量来自EV的散射强度和荧光强度，并在单颗粒水平上建立关联。标记的HD样品不仅显示出较低的荧光和散射强度，还显示出较低的有效折射率（电动汽车的散射和荧光强度可以同时测量，并在单粒子水平上相关。标记的HD样品不仅显示出较低的荧光和散射强度，还显示出较低的有效折射率（电动汽车的散射和荧光强度可以同时测量，并在单粒子水平上相关。标记的HD样品不仅显示出较低的荧光和散射强度，还显示出较低的有效折射率（Ñ ≈1.35）与LD电动汽车（比较Ñ ≈1.38），表明脂质和蛋白质二者内容物确实在HD电动汽车降低。尽管大小相似的电动汽车的密度梯度分离通常与生物分子含量的差异有关，但我们根据这些观察结果建议，分离反而反映了梯度溶质穿透包围电动汽车的脂质膜的能力，即两个梯度带更可能是因为膜的通透性差异，而不是电动汽车的生物分子含量差异。

更新日期：2018-06-20

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