Expansion Microscopy Of Tumor Cell Exosomes

Tumor cell exosomes are intraluminal vesicles secreted by tumor cells,which carry nucleic acids,proteins and other biologically active substances.They play a very important role in the process of tumor cell proliferation and metastasis.However,due to the nano-scale size and high heterogeneity of tumor cell exosomes,in-depth understanding of their appearance and biological characteristics is still lacking.Expansion microscopy(Ex M)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 Ex M,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,due to the small size of exosomes,Ex M or SMLM still cannot provide sufficient resolution.Therefore,this paper proposes a tumor cell exosomes imaging method that combines Ex M and SMLM(i.e.Expansion SMLM,denoted as Ex SMLM),which can realize the expansion and super-resolution imaging of tumor cell exosomes with a spatial resolution of <10 nm.Similar to SMLM,the imaging quality of Ex SMLM greatly depends on the optical performance of the fluorescent probes.We first proposed a super-resolution imaging probe based on ultrasmall silica nanospheres for SMLM imaging of tumor cell exosomes.The superresolution probe was fabricated by attaching fluorophores to ultrasmall silica nanospheres.It showed excellent characteristics suitable for long-term SMLM imaging,such as continuous fluorescence switching behavior and better localization precision(20-25 nm)than the fluorophore itself.In the experiment,we attached the human epidermal growth factor receptor2(HER2)aptamer to the super-resolution imaging probe,and successfully achieved precise localization and SMLM super-resolution imaging of HER2 overexpressed tumor cell exosomes.Subsequently,we studied the Ex SMLM technique of tumor cell exosomes.In this technique,we first used immunofluorescence to fluorescently label the protein markers on the tumor cell exosomes,and then polymerized the exosomes into a swellable polyelectrolyte gel.The hydrophilic nature of the gel made the fluorescently labeled exosomes undergo isotropic linear physical expansion.The expansion factor obtained in the experiment was 4.2.We further performed SMLM imaging of the expanded exosomes.The spatial resolution of tumor cell exosomes was improved to 7 nm.In conventional Ex M experiments,the loss and damage of fluorescent dyes will reduce the final image quality.We subsequently used ultrasmall silica nanoprobes instead of fluorescent antibodies in Ex SMLM to effectively improve the imaging quality of tumor cell exosomes.In summary,by expanding first and then performing SMLM imaging,the Ex SMLM technology can greatly improve the imaging resolution of tumor cell exosomes.This provides an important new technical means for the visualization of tumor cell exosomes-related disease mechanisms.