We have developed an accurate and practical method for measuring cytoplasmic [Ca2+] in the light microscoope. The method involves the use of the calcium-sensitive fluorescent dye Quin 2. The ratio of fluorescence intensities with excitation at 340 nm and 360 nm (I340/I360) provides a measure of [Ca2+] which is independent Quin 2 concentration. Using image intensification fluorescence microscopy and digital analysis of video images we are able to measure I340/I360 (and cytoplasmic [Ca2+]) throughout the cell. The video image analysis system provides enough temporal resolution, spatial resolution, and sensitivity to detect [Ca2+] gradients within a single cell using Quin 2. First, we will refine methods for measurement of localized [Ca2+]. We will then use the method to determine whether significant [Ca2+] gradients are developed in various cell types under a variety of physiological conditions. We have already found that mitotic cells have a considerable local variation in cytoplasmic [Ca2+]. Cell types to be studied including macrophages during phagocytosis, rat basophilic leukemia cells with clustered IgE receptors, and neutrophils exposed to chemotactic peptide. We will examine the contributions of ion pumping, diffusion, and buffering in dissipating [Ca2+] gradients, and we will try to obtain data on the sites of Ca2+ release into the cytoplasm. The role of intracellular messengers for the release of Ca2+ will be studied by micro-injecting putative messengers into the cytoplasm of Quin 2-loaded cells and observing changes in I340/I360. These studies will complement a large number of studies from several laboratories which are providing evidence for overall changes in cytoplasmic [Ca2+] throughout the cells. Local [Ca2+] gradients may be important for localized cell responses to external stimuli or for local control of the cytoskeleton.
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