4.7 Article

Nanoscale imaging of tumor cell exosomes by expansion single molecule localization microscopy (ExSMLM)

Journal

TALANTA
Volume 261, Issue -, Pages -

Publisher

ELSEVIER
DOI: 10.1016/j.talanta.2023.124641

Keywords

Exosomes; Expansion microscopy; Single molecule localization microscopy; Nanoscale substructures

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Tumor cell exosomes play a vital role in tumor cell proliferation and metastasis, but we still lack a comprehensive understanding of their appearance and biological characteristics due to their nanoscale size and high heterogeneity. We propose an imaging method called Expansion SMLM (ExSMLM) that combines Expansion Microscopy (ExM) and Single Molecule Localization Microscopy (SMLM) to achieve the expansion and super-resolution imaging of tumor cell exosomes.
Tumor cell exosomes play a very important role in the process of tumor cell proliferation and metastasis. However, due to the nanoscale size and high heterogeneity of exosomes, in-depth understanding of their appearance and biological characteristics is still lacking. Expansion microscopy (ExM) is a method that embeds biological samples in a swellable gel to physically magnify the samples to improve the imaging resolution. Before the emergence of ExM, scientists had invented several super-resolution imaging techniques that could break the diffraction limit. Among them, single molecule localization microscopy (SMLM) usually has the best spatial resolution (20-50 nm). However, considering the small size of exosomes (30-150 nm), the resolution of SMLM is still not high enough for detailed imaging of exosomes. Hence, we propose a tumor cell exosomes imaging method that combines ExM and SMLM (i.e. Expansion SMLM, denoted as ExSMLM), which can realize the expansion and super-resolution imaging of tumor cell exosomes. In this technique, immunofluorescence was first performed to fluorescently label the protein markers on the exosomes, then the exosomes were polymerized into a swellable polyelectrolyte gel. The electrolytic nature of the gel made the fluorescently labeled exosomes un-dergo isotropic linear physical expansion. The expansion factor obtained in the experiment was about 4.6. Finally, SMLM imaging of the expanded exosomes was performed. Owing to the improved resolution of ExSMLM, nanoscale substructures of closely packed proteins were observed on single exosomes, which has never been achieved before. With such a high resolution, ExSMLM would have a great potential in detailed investigation of exosomes and exosome-related biological processes.

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