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Optical Engineering

Combined analysis of intracellular calcium with dual excitation fluorescence photometry and imaging
Author(s): Dietmar Uttenweiler; Reinhold Wojciechowski; Makoto Makabe; Claudia Veigel; Rainer H.A. Fink
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Paper Abstract

We have developed an integrated microscopy system combining fast dual-excitation fluorescence photometry and digital image analysis with high spatial resolution, based mainly on standard components. With the combination of these well-established techniques in one setup it is possible to monitor intracellular calcium with both sufficiently high temporal and high spatial resolution on the same preparation for many biological applications. Our system consists of a commercially available dual-excitation photometric system, an attached intensified charge coupled device (ICCD) camera, and a frame grabber board. With this integrated setup one can easily switch between the fast photometric mode (vratio = 100 Hz) and the imaging mode (vratio = 4.l7 up to 17 Hz). We used the system to record Fura-2 calcium images (340/380 nm ratios), which were correlated with the faster spot measurements and were analyzed by means of image processing. As an example for its application we reconstructed caffeine-induced calcium transients released from the sarcoplasmic reticulum of isolated and permeabilized skeletal muscle fiber preparations. Such a combined technique will also be important for cellular studies using other fluorescence indicators. Additionally, the described system has an external trigger facility that enables combination with other cell physiological methods, e.g., electrophysiological techniques.

Paper Details

Date Published: 1 October 1995
PDF: 8 pages
Opt. Eng. 34(10) doi: 10.1117/12.210736
Published in: Optical Engineering Volume 34, Issue 10
Show Author Affiliations
Dietmar Uttenweiler, Univ. Heidelberg (Germany)
Reinhold Wojciechowski, Univ. Heidelberg (Germany)
Makoto Makabe, Univ. Heidelberg (Germany)
Claudia Veigel, Univ. Heidelberg (Germany)
Rainer H.A. Fink, Univ. Heidelberg (Germany)

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