title: Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro.
abstract: BACKGROUND
  Early and correct diagnosis of delayed or absent corneal epithelial wound healing is a key factor in the prevention of infection and consecutive destruction of the corneal stroma with impending irreversible visual loss. Two-photon microscopy (TPM) is a novel technology that has potential to depict epithelial cells and to evaluate cellular function by measuring autofluorescence properties such as fluorescence intensity and fluorescence lifetimes of metabolic co-factors such as NAD(P)H.
  
  
  METHODS
  Using non-invasive TPM in a tissue-culture scratch model and an organ-culture erosion model, fluorescence intensity and fluorescence lifetimes of NAD(P)H were measured before and during closure of the epithelial wounds. Influence of temperature and selective inhibition of metabolism on intensity and lifetimes were tested additionally.
  
  
  RESULTS
  Decrease of temperature resulted in significant increase of fluorescence lifetimes and decrease of the relative amount of free NAD(P)H due to decreased global metabolism. Increase in temperature and upregulation of glycolysis through blocking the mitochondrial electron transport chain by rotenone resulted in increased intensity, decreased lifetimes and increase in the relative amount of free NAD(P)H. Changes of lifetimes and free:protein-bound NAD(P)H ratios were similar to changes measured during wound healing in both scratch and erosion models.
  
  
  CONCLUSIONS
  Fluorescence lifetime measurements (FLIM) detected enhancement of cellular metabolism following epithelial damage in both models. The prospective detection of cellular autofluorescence in vivo, in particular FLIM of metabolic cofactor NAD(P)H, has the potential to become an indispensible tool in clinical use to differentiate healing from non-healing epithelial cells and to evaluate effects of newly developed substances on cellular metabolism in preclinical and clinical trials.
authors.affiliation: Department of Ophthalmology, University Hospital of Cologne, Kerpenerstr. 62, 50937 Cologne, Germany.
authors.forename: Uta
authors.initials: U
authors.lastname: Gehlsen
authors.forename: Andrea
authors.initials: A
authors.lastname: Oetke
authors.forename: Márta
authors.initials: M
authors.lastname: Szaszák
authors.forename: Norbert
authors.initials: N
authors.lastname: Koop
authors.forename: Friedrich
authors.initials: F
authors.lastname: Paulsen
authors.forename: Andreas
authors.initials: A
authors.lastname: Gebert
authors.forename: Gereon
authors.initials: G
authors.lastname: Huettmann
authors.forename: Philipp
authors.initials: P
authors.lastname: Steven
mesh_terms: D000818:Animals; D002057:Burns, Chemical / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005126:Eye Burns / Q000139:chemically induced* / Q000473:pathology; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D036641:Microscopy, Fluorescence, Multiphoton; D009249:NADP / Q000378:metabolism; D009924:Organ Culture Techniques; D012972:Sodium Hydroxide; D041623:Tomography, Optical Coherence; D014945:Wound Healing / Q000502:physiology*
chemical_list: D009249:NADP; D012972:Sodium Hydroxide