These results indicate that while CD63, CD81, and CD9 are all considered common exosome markers, many seminal exosomes express only one or two of these markers, and there is fantastic heterogeneity in tetraspanin expression levels both between exosomes and between these tetraspanins. == Number 2. by a new class of transistor-like, photoswitching Pdots. == Intro == Exosomes are lipid bilayer-enclosed nanoparticles that are secreted by cells and consist of biological cargo such as lipids, proteins, DNA, and RNA.[1]Intercellular communication via exosomes is usually thought to play a role in the pathogenesis of malignancy and inflammatory diseases.[2]Exosome surface proteins are key players in exosome biogenesis[1b,3]and contain information about the cell of origin of exosomes which can be useful in disease diagnosis.[4]To better understand exosome function, it is critical to obtain detailed information about surface proteins, such as copy number, spatial distribution and interactions between various types of proteins. However, there is currently a lack of tools for such studies. The small size and relatively low protein content IgG2a Isotype Control antibody (FITC) of exosomes make them difficult to become characterized by standard circulation cytometry.[5]Electron microscopy can reveal exosome structure, but is low-throughput and expensive. [6]Single-molecule imaging and superresolution microscopy are encouraging tools for characterizing biological constructions, [7]but also has low throughput compared with circulation cytometry. Here, we developed a high-throughput circulation method with single-molecule sensitivity for counting exosome surface proteins and for identifying exosome subtypes, followed by superresolution imaging analysis using a novel transistor-like semiconducting polymer dots (Pdots) for structural characterization and validation of the flow results. For the flow method, a microfluidic Crovatin platform was developed based on a line-confocal design,[7b]which consisted of four spatially-separated lasers lines, five detectors, and a custom-built autofocusing system. For flow analysis Crovatin of exosome size and surface protein copy number, exosomes are stained with a membrane dye and with fluorophore-conjugated antibodies. Depending on the flow rate, exosome concentration, and dye brightness, the flow system is usually capable of detecting hundreds to thousands of exosomes per second with single-molecule sensitivity. The fluorescence intensity of the membrane dye-stained exosomes is usually proportional to the surface area of the Crovatin lipid membrane,[8]allowing determination of exosome size. Protein copy number distributions are measured by deconvolving the intensity distributions of antibody-labeled exosomes using single antibody intensity distributions.[7a,7c]Using seminal exosome as a model, we performed profiling of three tetraspanins found on these exosomesCD63, CD81 and CD9, and decided their average copy number to be 12.8, 1.6, and 17.0, respectively. The heterogeneity in tetraspanin expression levels presented a challenge for single-molecule localization type of superresolution imaging as it is usually difficult to achieve both high throughput and high imaging quality.[9]To address this problem, we designed a novel class of photoswitching Pdots based on the theory of N-P-N transistors, which offers adjustable switch-on frequency based on the protein expression level and high localization precision. The Pdots exhibit spontaneous blinking and photoactivation in response to excitation at 405 nm, allowing the imaging duty cycle to be adjusted by over two orders of magnitude. Multi-color superresolution mapping of tetraspanins was performed by Crovatin using a combination of two Pdots and one fluorophore conjugated to antibodies against the three Crovatin tetraspanins. The duty cycle of the Pdots was adjusted based on tetraspanin copy numbers from flow analysis so that superresolution images of hundreds of exosomes could be obtained within five minutes, allowing resolution of the hollow structure of the exosomes and the spatial distributions of the tetraspanins with high precision. From the image analysis, we estimated the average spacing of CD63, CD81 and CD9 to be 39 nm, 122.