Imaging of hydrogen peroxide generation in cultured cells using dichlorofluorescein derivatives

Philip E. Hockberger; Mohammed S. Ahmed; Timothy A. Skimina; Christopher Lee; Wu-Yen Hung; Tippu Siddique

doi:10.1117/12.239502

10 May 1996 Imaging of hydrogen peroxide generation in cultured cells using dichlorofluorescein derivatives

Philip E. Hockberger, Mohammed S. Ahmed, Timothy A. Skimina, Christopher Lee, Wu-Yen Hung, Tippu Siddique

Proceedings Volume 2678, Optical Diagnostics of Living Cells and Biofluids; (1996) https://doi.org/10.1117/12.239502
Event: Photonics West '96, 1996, San Jose, CA, United States

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.

Citation Download Citation

Philip E. Hockberger, Mohammed S. Ahmed, Timothy A. Skimina, Christopher Lee, Wu-Yen Hung, and Tippu Siddique "Imaging of hydrogen peroxide generation in cultured cells using dichlorofluorescein derivatives", Proc. SPIE 2678, Optical Diagnostics of Living Cells and Biofluids, (10 May 1996); https://doi.org/10.1117/12.239502

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