Label-free identification of single cell-derived vesicles by Raman microspectroscopy

Type:
Oral presentation
Authors:
E. van der Pol, C.M. Hau, A.T.M. Lenferink, A. Sturk, R. Nieuwland, C. Otto, and T.G. van Leeuwen
Location: 
SPIE Photonics West 2013, San Francisco, United States
Date:
February 6, 2013

Summary

Human blood contains numerous cell-derived vesicles ranging from 30 nm to 1 μm in diameter. The function, origin, and composition of these vesicles is disease (state) dependent and therefore vesicles contain clinically relevant information. The cellular origin of vesicles is usually established by fluorescent antibody labeling, which involves practical problems. We have applied Raman microspectroscopy to distinguish normal vesicles from tumor-derived vesicles in solution without labeling. Single optically trapped tumor-derived vesicles showed unique Raman transitions compared to normal vesicles. For the first time, single tumor-derived vesicles were distinguished from normal vesicles without labeling using Raman microspectroscopy.

Abstract

Background: Human blood contains cell-derived vesicles, which are spherical particles enclosed by a phospholipid bilayer. These vesicles originate from blood cells, bone marrow cells, stem cells, and endothelial cells, and their function, origin, and composition is disease (state) dependent. Therefore, vesicles can be potentially used for prognosis, therapy, and biomarkers for disease. However, due to their small size (30 nm – 1 μm), detection of vesicles is cumbersome. The cellular origin of vesicles is usually established by fluorescent antibody labeling, which is laborious, expensive, and involves practical problems. We have applied Raman microspectroscopy to distinguish normal vesicles from tumor-derived vesicles in solution without labeling.

Methods: Platelet and erythrocyte vesicles were isolated from blood bank concentrates and tumor-derived vesicles were isolated from a human pancreatic adenocarcinoma cell line. Vesicles were isolated using differential centrifugation and analyzed by transmission electron microscopy, resistive pulse sensing, and Raman microspectroscopy. For Raman microspectroscopy, a 100-mW krypton ion laser operating at a wavelength of 647 nm was focused to a probe volume of 1 fL, which overlaps with the dimension of vesicles. The Stokes shift from light scattered by optically trapped vesicles was measured using a spectrograph dispersing in the range 646–849 nm.

Results: The Raman spectra of single optically trapped vesicles showed spectral transitions characteristic of phospholipids. Erythrocyte vesicles exhibited more fluorescence compared to platelet vesicles, whereas tumor-derived vesicles showed additional Raman peaks compared to normal vesicles.

Conclusions: For the first time, single tumor-derived vesicles were distinguished from normal vesicles without labeling using Raman microspectroscopy.

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