Label-free identification of extracellular vesicles by Raman microspectroscopy
||E. van der Pol, C.M. Hau, C. Otto, A.T.M. Lenferink, A. Sturk, T.G. van Leeuwen, and R. Nieuwland|
||International Society for Extracellular Vesicles 2012, Gothenburg, Sweden|
||April 19, 2012|
Background: The cellular origin of extracellular vesicles is usually established by fluorescent antibody labeling, which is laborious, expensive, and involves practical problems. Using an ultra-modern Raman microspectroscopy setup, we have identified extracellular vesicles without fluorescent antibody labeling.
Methods: Platelet and erythrocyte vesicles were isolated from blood bank concentrates. In addition, tumor-derived vesicles were isolated from BXPC3 and SIHA cell lines. Vesicles were isolated using differential centrifugation and analyzed by flow cytometry, transmission electron microscopy, Raman microspectroscopy, and resistive pulse sensing. For Raman microspectroscopy, a 100 mW krypton ion laser operating at a wavelength of 647 nm was focused to a probe volume smaller than 1 μm3. The Stokes shift from light scattered by vesicles was measured using a spectrograph dispersing in the range 646–849 nm. Resistive pulse sensing is a novel method capable of measuring the diameter, concentration, and surface charge of single vesicles in suspension using a nanopore.
Results: The Raman spectrum of vesicles with a different cellular origin is assessed. Platelet vesicles have a Raman spectrum containing spectral transitions characteristic of phospholipids. However, the Raman spectrum of for instance erythrocyte vesicles is different.
Conclusions: For the first time, vesicles are identified without fluorescent antibody labeling.