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Proceedings Paper

Imaging of hydrogen peroxide generation in cultured cells using dichlorofluorescein derivatives
Author(s): Philip E. Hockberger; Mohammed S. Ahmed; Timothy A. Skimina; Christopher Lee; Wu-Yen Hung; Tippu Siddique
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Paper Abstract

There is growing evidence that generation of reactive oxygen molecules (e.g., hydrogen peroxide, superoxide, hydroxyl and nitric oxide radicals) plays an important role in cell death. In this report we evaluated the effectiveness of the membrane permeable probe carboxy-dichlorodihydrofluorescein diacetate acetoxymethyl ester (C-DCDHF-DA-AM) for imaging the production of H2O2 in cultured cells. We examined the properties of three derivatives of the ester in saline droplets and compared the results with responses recorded from cells loaded with the ester. Results indicated that fluorescence was generated in cells and droplets by a photo-oxidative process involving H2O2. Videomicroscopy demonstrated that the cellular responses originated in small vesicles (presumably peroxisomes), with large responses filling the cytosol and enveloping the nucleus. We interpreted these responses as due to light-induced activation of flavin-containing oxidases, which generate H2O2 in peroxisomes, followed by diffusion of H2O2 throughout the cell. Escape of H2O2 from peroxisomes into Fe2+-containing compartments could have dire consequences on cell viability due to the production of hydroxyl free radicals. Such a mechanism could underly the phototoxic effects of visible light on cultured cells.

Paper Details

Date Published: 10 May 1996
PDF: 12 pages
Proc. SPIE 2678, Optical Diagnostics of Living Cells and Biofluids, (10 May 1996); doi: 10.1117/12.239502
Show Author Affiliations
Philip E. Hockberger, Northwestern Univ. Medical School (United States)
Mohammed S. Ahmed, Northwestern Univ. Medical School (United States)
Timothy A. Skimina, Northwestern Univ. Medical School (United States)
Christopher Lee, Northwestern Univ. Medical School (United States)
Wu-Yen Hung, Northwestern Univ. Medical School (United States)
Tippu Siddique, Northwestern Univ. Medical School (United States)

Published in SPIE Proceedings Vol. 2678:
Optical Diagnostics of Living Cells and Biofluids
Daniel L. Farkas; Robert C. Leif; Alexander V. Priezzhev; Toshimitsu Asakura; Bruce J. Tromberg, Editor(s)

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