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

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