Spatial Control of Reactive Oxygen Species Formation in Fibroblasts Using Two-photon Excitation

Authors

  • Brett A. King,

    1. Department of Dermatology, University of California, San Francisco, CA, Dermatology Research Unit, San Francisco Veterans Affairs Medical Center, San Francisco, CA
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    • Current address: Yale University School of Medicine, New Haven, CT.

  • Dennis H. Oh

    Corresponding author
    1. Department of Dermatology, University of California, San Francisco, CA, Dermatology Research Unit, San Francisco Veterans Affairs Medical Center, San Francisco, CA
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  • Posted on the website on 28 May 2004.

  • Current address: Yale University School of Medicine, New Haven, CT.

*To whom correspondence should be addressed: Dermatology Service (190), San Francisco VA Medical Center, 4150 Clement Street, San Francisco, CA 94121. Fax: 415-751-3927; e-mail: dennisoh@itsa.ucsf.edu

ABSTRACT

Two-photon excitation (2PE) provides a means of generating reactive oxygen species (ROS) in cells and tissues with a high degree of spatial specificity. In cultured monolayers of human fibroblasts and fibroblast-derived cells treated with the commonly used probe of ROS formation, 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), cells irradiated through a microscope objective with 150 fs near-infrared laser pulses became highly fluorescent, reflecting intracellular ROS formation. The fluorescence intensity inside cells increased quadratically with the average power of radiation for pulsed excitation and was unchanged for continuous wave irradiation with the same average power of radiation for pulsed excitation and was unchanged for continuous wave irradiation with the same average power. Single fibroblasts embedded within dermal equivalents were also targeted in this manner and formed ROS, whereas neighboring unirradiated cells were spared. These results demonstrate that ROS can be generated intracellularly in skin cells using 2PE of the metabolic or oxidative products of CM-H2DCFDA and that formation of ROS can be localized in both cell monolayers and in a tissue equivalent. This technique should be useful in understanding the response of whole tissues such as skin to local generation of ROS and may have applications in photodynamic therapy.

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