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Imaging and Intracellular Tracking of Cancer Derived Exosomes Using Single Molecule Localization Based Super Resolution Microscope Chen Chen, Shenfei Zong, Zhuyuan Wang, Ju Lu, Dan Zhu, Yizhi Zhang, and Yiping Cui ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09442 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016
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ACS Applied Materials & Interfaces
Imaging and Intracellular Tracking of Cancer Derived Exosomes Using Single Molecule Localization Based Super Resolution Microscope Chen Chen, Shenfei Zong, Zhuyuan Wang, Ju Lu, Dan Zhu, Yizhi Zhang, Yiping Cui*
Advanced Photonics Center, Southeast University, Nanjing 210096, China *
[email protected] Abstract Exosomes are small membrane vesicles secreted by cells and enriched with plenty of proteins. Considering their significant roles in different physical activities and potential value for diagnostic drug delivery, researchers have put great efforts in in-vitro tracking and contents analysis of exosomes. Recently, the emergence of different kinds of super-resolution microscopy provides powerful tools for exosome study. Here, we demonstrate the application of single molecule localization based super resolution imaging technique (PALM/STORM) in the imaging and tracking of cancer-derived exosomes. In the experiment, first, cancer-derived exosomes are extracted from the culture media of tumor cells. Then the exosome membrane receptors are labeled with photoswitchable probes, which allow super resolution imaging of these membrane receptors via photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). By using human breast cancer cell derived exosomes, we demonstrated simultaneous dual-color PALM/STORM imaging of two kinds of membrane receptors on the exosome membrane. Moreover, the successful labeling and imaging of exosomes make it possible to observe the interaction between cancer-derived exosomes and normal cells.
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Meanwhile, we realized the colocalization of cancer-derived exosomes and lysosomes in recipient cells with PALM/STORM imaging. Since exosomes play a vital role in intercellular communications, we anticipate that the presented PALM/STORM based imaging and tracking of exosomes holds a great potential in the investigation of the mechanism of exosome mediated cancer metastasis. Keywords:
exosomes,
membrane
receptors,
super-resolution
imaging,
PALM/STORM, intracellular tracking
1. Introduction Exosomes are small extracellular vesicles (EVs) with a diameter of 30–100 nm. Exosomes are secreted by a variety of cultured cells upon fusion of the multivesicular bodies (MVB) with the cell membrane1-4. Because of their origin, exosomes are enriched with a subset of proteins including members of the tetraspanin family (CD9, CD63 and CD81), members of the endosomal trafficking proteins (ESCRT-related proteins/Alix) and heat-shock proteins Hsp60, Hsp70 and Hsp702, 5. These small vesicles represent an important manner for the intercellular communication and exchanging of substances (such as delivery of cytosolic proteins, lipids and nucleic acids) without direct cell-to-cell contact3,
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and are potential for some practical
clinical application (e.g. diagnosis and drug delivery)10-11. For centuries, optical microscopy has greatly facilitated our understanding of molecular and cellular biology as well as the investigation of exosomes12. Compared with other imaging techniques,
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the outstanding advantage of fluorescence microscopy is its compatibility with samples, which ensures noninvasive imaging and high biochemical specificity13.
However, the moderate resolution of several hundred nanometers for conventional optical microscopy limits the accurate localization and imaging of exosomes considering their relatively small size (
