Observation of Insulin Exocytosis by a Pancreatic β Cell Line with Total Internal Reflection Fluorescence Microscopy

INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.

Three-dimensional tracking of GLUT4 vesicles in TIRF microscopy

TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.

Practical applications of total internal reflection fluorescence microscopy for nanocatalysis

Fluorescence microscopy has evolved from a purely biological tool to a powerful chemical instrument for imaging and kinetics research into nanocatalysis.And the demand for high signal-to-noise ratio and temporal–spatial resolution detection has encouraged rapid growth in total internal reflection fluorescence microscopy(TIRFM).By producing an evanescent wave on the glass–water interface,excitation can be limited to a thin plane to ensure the measured accuracy of kinetics and image contrast of TIRFM.Thus,this unique physical principle of TIRFM makes it suitable for chemical research.This review outlines applications of TIRFM in the field of chemistry,including imaging and kinetics research.Hence,this review could provide guidance for beginners employing TIRFM to solve current challenges creatively in chemistry.

RAB-27 and its effector RBF-1 regulate the tethering and docking steps of DCV exocytosis in C.elegans

The molecular mechanisms by which dense core vesicles(DCVs) translocate,tether,dock and prime are poorly understood.In this study,Caenorhabditis elegans was used as a model organism to study the function of Rab proteins and their effectors in DCV exocytosis.RAB-27/AEX-6,but not RAB-3,was found to be required for peptide release from neurons.By analyzing the movement of DCVs approaching the plasma membrane using total internal reflection fluorescence microscopy,we demonstrated that RAB-27/AEX-6 is involved in the tethering of DCVs and that its effector rabphilin/RBF-1 is required for the initial tethering and subsequent stabilization by docking.