We have developed an imaging approach to estimate the diameter of exocytic vesicles that are smaller than the resolution of an optical microscope and present within intact tissue. This approach is based on two-photon excitation imaging of polar tracers in the extracellular medium, is designated TEPIQ (two-photon extracellular polar-tracer imaging-based quantification), and has three variants. TEPIQ analysis of ΔV measures vesicle volume with a fluid-phase tracer, sulforhodamine B (SRB). TEPIQ analysis of ΔS determines vesicle surface area with a polar membrane tracer, FM1-43. TEPIQ analysis of ΔV/ΔS estimates vesicle diameter from the SRB/FM1-43 fluorescence ratio. TEPIQ analysis is insensitive to microscope settings because the same setup is used for calibration and actual experiments. We tested the validity of TEPIQ with glucose-induced exocytosis from beta-cells within pancreatic islets. The three TEPIQ variants yielded estimates for the mean diameter of exocytic vesicles; of between 340 and 390 mn, consistent with the size of insulin granules. TEPIQ analysis relies on the combination of two-photon excitation imaging, the narrow intercellular spaces of intact tissue, and the presence of diffusible polar tracers in the extracellular medium. It allows quantitative imaging of exocytosis within secretory organs, yielding estimates of vesicle diameter with nanometer resolution.
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