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Proceedings Paper

Instrumentation for simultaneous kinetic imaging of multiple fluorophores in single living cells
Author(s): Stephen J. Morris; Diane M. Beatty; Larry W. Welling; Thomas B. Wiegmann
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Paper Abstract

Low-light fluorescence video microscopy has established itself as an excellent method for investigations of cell dynamics. There is a growing interest in resolving multiple images of 'ratio' fluorophores like indo or BCECF or the emission from multiple dyes placed in the same cell system. For rapid kinetic studies, the problems of photodynamic damage and photobleaching on one hand and the need for good spatial and temporal resolution on the other, press the resolution of the instrumentation. Rapid resolution of multiple probes at multiple wavelengths presents a third set of problems of exciting the probes and appropriately imaging the emitted light. The authors have designed a new real-time low-light fluorescence video microscope for capturing intensified images of up to four dyes contained in the same cell system. These can be two dual-emission wavelength 'ratio' dyes or multiple dyes. The optics allow simultaneous excitation of up to four fluorophores and the real-time (30 frames/second) capture of four separate fluorescence emission images. Each emission wavelength is imaged simultaneously by one of four cameras, then digitized and appropriately combined at standard video frame rates to be stored at high resolution on tape or video disk for further off-line correction and analysis. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in filter wheel or mechanical shutter designs for multiple imaging. The instrument can be assembled form off-the-shelf components. Coupled to compatible image processing software utilizing PC-AT computers, it can be realized for relatively low cost. Two examples of simultaneous multi-parameter imaging are presented. Synchronous observations of calcium and pH distribution in kidney epithelial cells, loaded with both indo-1 and SNARF-1TM, show that both are altered in response to ionomycin treatment; however, the kinetics for the two changes are quite different. Intracellular calcium increases rapidly when the bath Ca2+ is raised. The pH remains stable for several seconds, then suddenly collapses. The second example concerns fusion of human red blood cells (RBC) to fibroblasts expressing influenza hemagglutinin. Movement of soluble and membrane-bound dyes follow different kinetics, depending upon the molecular weight of the soluble dye. Furthermore, the swelling of the RBC occurs after the onset of fusion, and therefore cannot provide the driving force.

Paper Details

Date Published: 1 May 1991
PDF: 11 pages
Proc. SPIE 1428, Three-Dimensional Bioimaging Systems and Lasers in the Neurosciences, (1 May 1991); doi: 10.1117/12.44137
Show Author Affiliations
Stephen J. Morris, Univ. of Missouri/Kansas City (United States)
Diane M. Beatty, Univ. of Missouri/Kansas City (United States)
Larry W. Welling, Veterans Administration Medical Ctr. and Kansas Univ. Medical Ctr. (United States)
Thomas B. Wiegmann, Veterans Administration Medical Ctr. and Kansas Univ. Medical Ctr. (United States)


Published in SPIE Proceedings Vol. 1428:
Three-Dimensional Bioimaging Systems and Lasers in the Neurosciences
James E. Boggan; Leonard J. Cerullo; Louis C. Smith, Editor(s)

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