**1. Introduction**

Extracellular vesicles (EVs) are heterogeneous because of their diverse cell of origins, the process of biogenesis, the specific stimuli in their microenvironment and so on. EVs are produced by cells of bacteria, fungi, plants and animals. Inside human bodies, EVs carry molecular signatures of their parent cells and diffuse freely among blood stream and tissues. EVs can be classified into various subpopulations according to their origin, size, density, biogenesis, compositions etc. The origin of the vesicles gives us terminology of prostasome, oncosome etc. According to the size of EVs, investigators utilized terms of exomere, small EVs, large EVs. Depending on the biogenesis, we defined apoptic bodies, microvesicles and exosomes. Apoptic bodies are large vesicles formed due to apoptosis. Microvesicles are vesicles budding directly from cell membrane. Exosomes are the released intraluminal vesicles (ILVs) from multivesicular bodies (MVBs) through fusion of MVB with cell membrane.

To address the heterogeneity of EVs, scientists dedicated to the development of novel techniques for single EV detection. Besides investigation of EV biology, single EV analysis technologies are promising approach for liquid biopsy, which relies on the detection of biomarker EVs readily available in body fluids. Here in this chapter, we will discuss four main strategies of studying single EVs, including single EV imaging systems, flow cytometers, nano-sensing technologies and single EV barcoding assay.
