Quantitative analysis of diffraction-limited three-dimensional single secretory vesicle tracking in live neuroendocrine cells

Neuroendocrine cells release hormones and transmitters by regulated exocytosis of secretory vesicles. Three-dimensional (3D) tracking of single secretory vesicles throughout live whole cells is crucial for understanding the intracellular trafficking of secretory vesicles. But one notable problem with 3D particle tracking is that the lateral and axial resolution is not equivalent for 3D live-cell images, which is attributed to the diffraction limit inherent to the microscope. How the performance of particle tracking is affected by diffraction-limited images is still unclear. Here we show, employing both centroid and Gaussian-fit tracking algorithm to monitor sub-pixel positions of single fluorescent particles, that the performance of three-dimensional single particle tracking is nonisotropic. The efficacy of the particle tracking in the axial direction is much lower than that in the lateral direction. Gaussian-fit tracking algorithm provides better performance in the axial direction at low signal-to-noise levels around or below 10. Using neuroendocrine cell line PC12, we illustrate the feasibility of Gaussian-fit algorithm to follow the mobility of single secretory vesicles in all three dimensions.