title: FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol.
abstract: OBJECTIVE
  To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM).
  
  
  METHODS
  Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC.
  
  
  RESULTS
  During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR.
  
  
  CONCLUSIONS
  This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire, Pitié-Salpêtrière, Paris, France.
authors.forename: Edmond
authors.initials: E
authors.lastname: Kahn
authors.forename: Anne
authors.initials: A
authors.lastname: Vejux
authors.forename: Dominique
authors.initials: D
authors.lastname: Dumas
authors.forename: Thomas
authors.initials: T
authors.lastname: Montange
authors.forename: Frédérique
authors.initials: F
authors.lastname: Frouin
authors.forename: Vincent
authors.initials: V
authors.lastname: Robert
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Riedinger
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Stoltz
authors.forename: Philippe
authors.initials: P
authors.lastname: Gambert
authors.forename: Andrew
authors.initials: A
authors.lastname: Todd-Pokropek
authors.forename: Gérard
authors.initials: G
authors.lastname: Lizard
mesh_terms: D016923:Cell Death / Q000187:drug effects; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007653:Ketocholesterols / Q000633:toxicity*; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects; D009000:Monocytes / Q000737:chemistry / Q000187:drug effects*; D010078:Oxazines; D013194:Staining and Labeling; D020298:U937 Cells
chemical_list: D005456:Fluorescent Dyes; D007653:Ketocholesterols; D010078:Oxazines; C003001:7-ketocholesterol; C044808:nile red