**2.5 Stochastic optical reconstruction microscopy (STORM)**

Stochastic optical reconstruction microscopy (STORM) and direct-STORM (d-STORM) are single-molecule super-resolution imaging techniques with a practical resolution limit of 20 nm. STORM utilized the photoswitchable fluorescent probes to precisely localize detected molecules at a high spatial resolution. AlexaFluor 647-conjugated anti-CD63 antibodies were employed to detect cancer cell-derived EVs [9]. Using photo-switchable lipid dyes such as Dil, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size and imaging of EV uptake by live cells in culture [10]. With STORM, droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of EVs with cancer-specific biomarkers (**Figure 5**) [11].

#### **Figure 3.**

*TIRFM for single EV 3-plexed proteomic biomarker analysis. The colocalization of multiple biomarkers could be simultaneously recorded via multi-color TIR imaging.*

*High-Throughput Single Extracellular Vesicle Profiling DOI: http://dx.doi.org/10.5772/intechopen.97544*

**Figure 4.** *TIRFM for single EV miRNA biomarker analysis.*

#### **Figure 5.**

*Imaging of two adjacent exosomes with conventional TIRFM image, PALM/STORM image. Cross-sectional profiles of the two adjacent exosomes shown.*
