Live-cell imaging of vesicle trafficking and divalent metal ions by total internal reflection fluorescence (TIRF) microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy is an especially powerful tool for visualizing live cellular events. Fluorescent molecules alone provide broad information about the expression and localization of proteins and other molecules; however, the temporal and spatial resolution is confounded by signal from outside the area of interest and the intensity of the illumination required. TIRF overcomes this limitation by using the reflective properties of a laser beam to illuminate a narrow (<100 nm) strip at the surface of a cell with a relatively low powered evanescent wave, thus making it possible to measure events occurring specifically at the plasma membrane such as exocytosis, single molecule interactions, and ionic changes during signal transduction. Here we describe some of the methods for using TIRF microscopy to study the processes involved in exocytosis from excitable cells (i.e., neurons, endocrine, neuroendocrine, and exocrine cells) and the release of physiologically active substances (i.e., neurotransmitters, hormones, and mucus).The failure of regulated exocytosis is associated with various diseases such as allergy, brain dysfunction, and endocrine illness. Diabetes mellitus, which is due to an absolute (type I) or relative (type II) deficiency of insulin secretion from pancreatic β-cells, is a major area of therapeutic interest. Insulin is stored in dense core vesicles with Zn(2+) ions in pancreatic β-cells. Insulin secretion is regulated by plasma glucose concentration which acts through intracellular metabolism to influence intracellular [Ca(2+)]. However, the precise molecular mechanisms controlling insulin granule movement towards, and fusion at, the plasma membrane remain only partially understood. To tackle this problem, we have used live cell imaging techniques to image regulated exocytosis in single living β-cells alongside intracellular Ca(2+) and Zn(2+) concentrations.