Sub-Frame Time Resolution in Fluorescence Imaging Reveals Delay Between SNAP25 Conformational change and Secretory Events in Chromaffin Cells

Abstract

protein Von Willebrand factor (VWF), however, under specific conditions WPBs also contain a cocktail of small pro-inflammatory cytokines. WPB exocytosis is driven by an increase in intracellular free calcium ion concentration ([Ca 2þ ] i ); the majority of WPB fusion events result in complete discharge of cargo components, however, in a small fraction of cases cytokines are selectively released indicating that the fusion pore may act as a molecular size filter. Carbon fibre amperometry has been used to characterize fusion pore behavior in a number of cell types, but to date this approach has not been applied to WPBs. Therefore, we used this technique in combination with simultaneous optical imaging of fluorescent WPB exocytosis and changes in [Ca 2þ ] i and can report, for the first time, the kinetic properties of WPB fusion pore formation and expansion in human cultured endothelial cells. A clear delay (mean ~50 ms) is seen between the onset of the current spike and the increase in intra-WPB EGFP fluorescence indicating that WPB alkalinsation is delayed by the strong proton buffering capacity of the WPB lumen (55 mM/pH unit). Analysis of current spike parameters reveal a mean 25-75% rise time, peak amplitude and decay time of 1.63 ms, 50 pA and 6.63 ms respectively. Approximately 50% of current spikes were preceded by a foot signal of mean duration 6.34 ms. Occasional low amplitude, prolonged current increases, reminiscent of stand alone foot signals, were observed in conjunction with morphological rounding of WPBs, possibly reflecting kiss-and-run or lingering kiss fusion events. Following characterization of the WPB fusion pore under control conditions the impact of changing cellular parameters, including cholesterol levels, was also assessed.

Precise Time Superresolution by Event Correlation Microscopy

“…Such imaging experiments are frequently performed at very low intensity, which means that the rate of photon detection is low and camera exposure times are long such that signal averaging is used to obtain an acceptable SNR. We developed the ECOM method that aligns and averages the imaging data relative to an HTR event with a precision that is not limited by camera exposure time and applied it to analyze small FRET changes associated with individual transmitter release events detected as an amperometric spike that serves as the HTR event (7,8). Here, we provide a detailed theoretical, computational, and experimental analysis of the method and its precision in determination of the time of the intensity step with subimaging frame resolution.…”

“…When multiple events occurring at random times during an exposure are averaged, the time correlation between the physical events and the fluorescence change can be determined with subframe resolution. We previously applied ECOM to analyze small FRET changes associated with individual transmitter release events detected as an amperometric spike that serves as the HTR event (7,8). Here, we provide a detailed theoretical, computational, and experimental analysis of the method and its precision in determination of the time of the intensity step with subimaging frame resolution.…”