Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles

IF 15.5 1区 医学 Q1 CELL BIOLOGY Journal of Extracellular Vesicles Pub Date : 2024-10-14 DOI:10.1002/jev2.12512
Andreas Wallucks, Philippe DeCorwin-Martin, Molly L. Shen, Andy Ng, David Juncker
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Abstract

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7 min over ten 88 × 88 µm2 fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220 nm in diameter with a dual 440 and 740 nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100 nm-in-diameter EVs with fluorescently tagged membrane proteins.

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固定沉浸式细胞外囊泡的尺寸光度测量和荧光成像
在普通荧光显微镜下对单个细胞外囊泡 (EV) 进行免疫荧光分析因其易用性和高荧光灵敏度而越来越受欢迎;然而,EV 的数量和大小只能通过荧光染色剂来测量,需要对样品进行大量操作。在这里,我们介绍了基于干涉散射(iSCAT)成像的高灵敏度无标记 EV 粒度光度法(SP),该成像可对固定在玻璃盖玻片上的浸泡 EV 进行测量。我们在普通的倒置外荧光显微镜上使用 LED 照明和简单的 50:50 分光镜实现了 SP,从而实现了 SP 与荧光成像(SPFI)的无缝集成。我们介绍了一种高通量 SPFI 工作流程,可在 7 分钟内在 10 个 88 × 88 µm2 视场中记录 10,000 个 EV,并在孵育前后成像以抑制背景,同时自动进行图像对齐、像差校正、光斑检测和 EV 大小调整。我们采用 440 和 740 纳米双 SP 照明方案,实现了从直径 37 纳米到 ∼220 纳米的 EV 大小范围,并建议通过更先进的图像分析或额外的硬件定制来扩展这一范围。我们使用校准过的二氧化硅纳米粒子将 SP 与流式细胞仪进行了比对,结果表明 SPFI 具有卓越的无标记灵敏度。我们通过实验区分了表面和体积EV染料,观察了吸附在表面上的EV的变形,并发现了<100 nm直径EV中带有荧光标记膜蛋白的不同亚群,从而展示了SPFI在EV分析方面的潜力。
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来源期刊
Journal of Extracellular Vesicles
Journal of Extracellular Vesicles Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
27.30
自引率
4.40%
发文量
115
审稿时长
12 weeks
期刊介绍: The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies. The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.
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