Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation.

摘要: The trajectory of secretory vesicles to their fusion sites at the plasma membrane is expected give insight into mechanisms that underlie vesicle transport, maturation and initiation fusion. Evanescent-wave (EW) microscopy allows tracking fluorescently labeled granules prior with nanometer precision in xy-direction. At same time, exponential sensitivity granular fluorescence experimental parameters can preclude quantitative estimates granule's approach membrane. Thus, it has remained controversial which extent axial distance be obtained from simple intensity measurements. We used information contained a stack images acquired 80-125 nm penetration depth EW field estimate individual granule diameter distance. A population analysis on 90 revealed an average 305 +/- 47 nm, below diffraction-limited 352 31 xy measurements fixed penetration. Stimulation exocytosis by potassium depolarization resulted selective loss 18 5% located closest membrane, while second 60 deeper within cytoplasm increased recruitment previously > or = 120 depth. These extend corroborate previous observations functionally distinct populations. Parameters influencing accuracy parameter estimation are evaluated appendix.