**2.3 Microfluidic device for single EV imaging**

Combining with fluorescent microscopies, microfluidic devices are widely designed for single EV analysis. The EVs were biotinylated and captured on the streptavidin coated glass surface of a microfluidic device. The repeated cycles of immune staining-imaging-quenching steps allowed the investigators to analyze up to 11 biomarkers on immobilized individual EVs with an inverted microscope (Nikon Eclipse TE2000S) equipped with a sCMOS camera. After image processing, about 600 individual EVs were mapped onto a 2D plot via t-distributed stochastic neighbor embedding (tSNE) [6]. However, Biomarkers expressed on single EVs are at very

#### **Figure 2.**

*AFM for analysis of single EVs. a) Principles of AFM detection. Panel b) shows an typical AFM readout of EV morphology detection and particle size distribution plot. c) and d) AFM measurement of biomechanical properties of EVs.*

low abundant or has limited number of epitopes for immunostaining. Therefore, EV biomarker could be undetected due to weak signals. Molecular profiling of single EVs often requires high resolution optical microscopies or signal amplification strategies, such as rolling circle amplification, branched DNA probes or enzymatic reactions.
