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Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment
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vesicles immobilized via molecular linkers at a solid support represent a convenient platform for basic and applied studies of biological processes occurring at lipid
membranes. Using total internal reflection fluorescence microscopy (TIRFM), one can track such processes at the level of individual vesicles provided that they contain dyes. In such experiments, it is desirable to determine the size of each vesicle, which may be in the range from 50 to 1000 nm. Fortunately, TIRFM in combination with nanoparticle tracking analysis makes it possible to solve this problem as well. Herein, we present the formalism allowing one to interpret the TIRFM measurements of the latter category. The analysis is focused primarily on the case of unpolarized light. The specifics of the use of polarized light are also discussed. In addition, we show the expected difference in size distribution of suspended and immobilized vesicles under the assumption that the latter ones are deposited under diffusion-controlled conditions. In the experimental part of our work, we provide representative results, showing explicit advantages and some shortcomings of the use of TIRFM in the context under consideration, as well as how our refined formalism improves previously suggested approaches.
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