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Proceedings Paper

Fluorescence Ratio Imaging Of Dynamic Intracellular Signals
Author(s): A. T. Harootunian; J. P.Y. Kao; Roger Y. Tsien
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Paper Abstract

Traditional biochemical assays of cellular messengers require grinding up thousands or millions of cells for each data point. Such destructive measurements use up large amounts of tissue, have poor time resolution, and cannot assess heterogeneity between individual cells or dynamic spatial localizations. Recent technical advances now enable important ionic signals to be continuously imaged inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+. Binding of these ions shifts the fluorescence excitation spectrum of the corresponding indicator. The ratio of excitation amplitudes at two wavelengths measures the free ion concentration while canceling out intensity variations due to nonuniform cell thickness or dye content. By rapidly alternating between the two ion-sensitive excitation wavelengths, a fluorescence microscope equipped with a low-light television camera and digital image processor can produce dynamic images of intracellular messenger levels. In many populations of cells traditionally assumed to be homogeneous, we find that neighboring individual cells can differ enormously in their cytosolic Ca2+ response to agonist stimulation, some ignoring the stimulus, others raising cytosolic Ca2+ transiently, others showing oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of graded inputs; we are investigating the mechanism of their generation using light flashes to generate pulses of intracellular messengers. Spatial gradients of cytosolic Ca t+ within single cells have been observed in embryos during fertilization and development, neurons exposed to electrical or drug stimulation and in cytotoxic T lymphocytes during killing of target cells. Variations of cytosolic pH and free Na+ are especially interesting in epithelia and in cells undergoing mitogenic or oncogenic activation.

Paper Details

Date Published: 22 December 1989
PDF: 5 pages
Proc. SPIE 1161, New Methods in Microscopy and Low Light Imaging, (22 December 1989); doi: 10.1117/12.962706
Show Author Affiliations
A. T. Harootunian, University of California, Berkeley (United States)
J. P.Y. Kao, University of California, Berkeley (United States)
Roger Y. Tsien, University of California, Berkeley (United States)

Published in SPIE Proceedings Vol. 1161:
New Methods in Microscopy and Low Light Imaging
John E. Wampler, Editor(s)

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