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identified and analyzed by making meas urements on a population of single enti ties, for example, single liposomes [4][5][6][7][8][9] or EVs. [10,11] Traditional assays that treat a population as an ensemble obscure the distributions of particle properties, and they are blind to the asynchronous events that are revealed at the single cell, particle or molecule level.Single entity measurements can be obtained using microscopy, [12][13][14][15] spec troscopy, [16,17] flow cytometry, [10,18] mass spectrometry, [19,20] electrical, [21] electro chemical, [22] and separation methods, [23] all of which can reveal particle heteroge neities. However, many of these methods have weaknesses that limit their appli cability. For example, electron micro scopy has the requisite spatial resolution to observe individual small particles, but because of the required sample processing and fixing, temporal dynamics among particles cannot be determined. Flow cytometry is unable to characterize nanoscale (50-500 nm) objects, such as EVs, without special instrument modifications. Additionally, flow cytometry, capillary electrophoresis, and elec trochemical cytometry have highthroughput capabilities, but they are unable to analyze a single isolated particle over long periods of time, and specific individual particles are gener ally not recoverable for further analysis. Also, some of these methods destroy the particles as part of the measurement pro cess, which prevents multiplex analysis of individual particles. To overcome these drawbacks, new approaches are necessary that leverage the strengths of optical microscopy and enable multiplex highthroughput measurements on single biological particles to reveal their physical, chemical, and physiological heterogeneities.Chemicallyspecific capture of individual particles on sur faces can enable single particle analysis. [24,25] Furthermore, this approach can be improved by patterning the capture molecules in densely packed, highly ordered arrays, resulting in densely packed, highly ordered arrays of particles. [7] Pat terning the capture molecules on a surface results in the cap ture of thousands of particles in ordered arrays within a single image frame. Patterning capture molecules into highly ordered arrays ensures that neighboring particles are optically resolv able, while at the same time particle surface density is maxi mized. Additionally, capture molecule nanodots that are similar Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access ...

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

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