Serum-derived exosomes are membrane-enclosed nanovesicles secreted by cells, typically between 30 and 120 nm in diameter. Exosomes can be identified by the presence of tetraspanin protein markers including CD9, CD81, and CD63 among others. The relative amounts of these exosomal markers and their location in the exosomes are also related to their source of origin. The ability to investigate these different markers and their locations in individual and multiple exosomes was obtained using an optical imaging technique known as dSTORM (direct stochastic optical reconstruction microscopy) which can overcome the diffraction limit for detection of these nanovesicles. The use of the dSTORM imaging method has allowed us to evaluate the relative abundance of tetraspanin markers CD9, CD81, and CD63 in exosomes and the size of exosomes related to these markers. We also compared the presence of these markers in normal versus pancreatic cancer serum samples and against exosomes secreted from cell lines. We found that CD9 is generally the most abundant marker in exosomes and is found near the surface of the exosomes, although CD81 and CD63 abundance is also significant. This result is consistent with our prior DIA mass spectrometry data and may be important in future work involving analysis of exosomes as markers of disease.