Two-Photon Fluorescence Microscopy Imaging of Cellular Oxidative Stress Using Profluorescent Nitroxides
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- 作者：Hyo-Yang Ahn ; Kathryn E. Fairfull-Smith ; Benjamin J. Morrow ; Vanessa Lussini ; Bosung Kim ; Mykhailo V. Bondar ; Steven E. Bottle ; Kevin D. Belfield
- 刊名：The Journal of the American Chemical Society
- 出版年：2012
- 出版时间：March 14, 2012
- 年：2012
- 卷：134
- 期：10
- 页码：4721-4730
- 全文大小：596K
- 年卷期：v.134,no.10(March 14, 2012)
- ISSN：1520-5126
文摘
A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore鈥檚 usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H2O2-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially 鈥渓ight up鈥?in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.