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A new quantitative (two‐photon extracellular polar‐tracer imaging‐based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets
Author(s) -
Kasai Haruo,
Hatakeyama Hiroyasu,
Kishimoto Takuya,
Liu TingTing,
Nemoto Tomomi,
Takahashi Noriko
Publication year - 2005
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2005.093047
Subject(s) - vesicle , exocytosis , sulforhodamine b , extracellular , biophysics , chemistry , secretory vesicle , two photon excitation microscopy , microscopy , pancreatic islets , membrane , optics , fluorescence , biology , physics , biochemistry , islet , insulin , in vitro , endocrinology , cytotoxicity
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 Δ V measures vesicle volume with a fluid‐phase tracer, sulforhodamine B (SRB). TEPIQ analysis of Δ S determines vesicle surface area with a polar membrane tracer, FM1‐43. TEPIQ analysis of Δ V /Δ S 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 nm, 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.