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P. Steven, A. Oetke, U. Gehlsen, R. Orzekowsky-Schroeder, N. Koop, M. Mueller, G. Huettmann; Non Invasive Two-Photon Fluorescence Lifetime Measurement (FLIM) Monitors Metabolic Changes During Wound Healing of Corneal Epithelial Cells in vitro . Invest. Ophthalmol. Vis. Sci. 2010;51(13):396. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Measurement of cellular metabolism during corneal epithelial wound healing requires tissue processing and ex-vivo analytical methods. Two-photon fluorescence lifetime measurement (FLIM) has been demonstrated to be a suitable non-invasive optical method to analyze consecutive changes in NADH metabolism in cancer cells in-vitro. In this study we used FLIM to analyze changes of cellular NADH-metabolism in tissue and organ cultures to investigate a potential use of two-photon FLIM as a future non-invasive optical device for analyzing metabolic changes during wound healing processes at the ocular surface in-vivo.
Non-invasive two-photon FLIM was conducted on a tissue-culture scratch model (HCE-cells) and a murine eye organ culture (NaOH-erosion). Changes of mean NADH-lifetimes and free to bound NADH ratios were analyzed in tissue and organ cultures before and directly after manipulation and repeatedly until closure of scratch or erosion. In addition, changes of mean NADH-lifetimes and NADH-ratios were investigated after application of the respiratory chain inhibitor rotenone. Data was analyzed by SPCImage software.
Mean NADH-lifetimes in HCE tissue cultures were appr. 1520ps. Reduction of cellular metabolism by inhibiting the respiratory chain resulted in dose dependent decrease of mean NADH-lifetimes to appr. 1300ps. Generation of scratches in HCE monolayers also resulted in immediate reduction of NADH-lifetimes, followed by increasing lifetimes during scratch closure. Accordingly, in organ cultures the generation epithelial erosion resulted in mean lifetime reduction followed by steady increase until closure of the wound. Free to bound NADH-ratios decreased after erosion, indicating an initial reduction of the protein-bound NADH proportion with consecutive restriction of cellular metabolism.
Two-photon FLIM allows to analyze changes in NADH metabolism of corneal epithelial cells during wound healing in vitro. These results suggest a potential use of two-photon FLIM for non-invasive intravital metabolic mapping of corneal epithelial cells in wound healing models and possible intravital detection of wound healing dysfunction due to chemical burns, stem cell deficiencies and surgical manipulation.
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