**2.4 Total internal reflection fluorescence microscopy (TIRFM)**

Total internal reflection fluorescence microscopy (TIRFM) is an optical technique utilized to observe single molecule fluorescence at surfaces and interfaces. It provides a high axial resolution below 100 nm. Single-vesicle imaging analysis could be done via TIRFM. The investigators can visualize multiple marker expressions of individual EVs by using fluorescent probes, and can also classify EV subpopulations by analyzing co-localization of markers [7]. With TIRFM system, targeted miRNA detection at single EVs could be realized with the co-delivery of inactive split DNAzymes and fluorescence-quenched substrates into EVs and a miRNA-activated catalytic cleavage reaction that amplifies fluorescence signal (**Figures 3** and **4**) [8].
