Refractive index determination of single sub micrometer vesicles in suspension using dark-field microscopy
||E. van der Pol, F.A.W. Coumans, A.N. Böing, A. Sturk, R. Nieuwland, T.G. van Leeuwen|
||SPIE Photonics West 2014, San Francisco, United States|
||February 1, 2014|
||San Fransisco 2014 Refractive index vesicles.pdf (1,229 kB)|
Cell-derived vesicles are abundantly present in blood and contain clinical information. Isolation of vesicles (30 nm – 1 μm) is cumbersome, since blood contains similar-sized particles, e.g. lipoproteins. The refractive index may provide a new label-free parameter to distinguish vesicles from lipoproteins. However, no method is available to determine the refractive index of single vesicles. We have measured the diameter and scattering of beads and vesicles by tracking their Brownian motion with dark-field microscopy and theoretically described the results to determine the refractive index of vesicles. We provide evidence that vesicles can be distinguished from lipoproteins based on their refractive index.
Background: Cells release vesicles, also called exosomes or microparticles, which are spherical particles containing a phospholipid bilayer. These vesicles are abundantly present in human blood and it is becoming increasingly clear that they contribute to many homeostatic processes, for example coagulation and inflammation. Therefore, vesicles are a potential biomarker for disease. Isolation and detection of vesicles is a major challenge, since vesicles are small (30 nm – 1 μm) and blood contains similar-sized particles, such as lipoproteins. The refractive index may provide a new label-free parameter to distinguish vesicles from lipoproteins. However, currently no method is available to determine the refractive index of single vesicles in suspension at high throughput.
Method: We have measured the diameter and light scattering of vesicles and beads of known properties by tracking their Brownian motion with dark-field microscopy (NS500, Nanosight Ltd). We analytically described the relation between the diameter, refractive index, and light scattering of beads by Mie theory to determine the refractive index of vesicles from urine and blood.
Results: We obtained a median refractive index of urine vesicles of 1.36, whereas the refractive index distribution of vesicles from blood was much broader and had a median refractive index of 1.49.
Conclusions: Dark-field microscopy can be used to assess the refractive index of single sub micrometer vesicles in suspension. Urine vesicles had a different median refractive index (n=1.36) than particles from plasma (n=1.49). We hypothesize that the relatively high refractive index of plasma particles is due to the presence of lipoproteins.