FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol.
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.
Kahn
Edmond
E
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire, Pitié-Salpêtrière, Paris, France.
Vejux
Anne
A
Dumas
Dominique
D
Montange
Thomas
T
Frouin
Frédérique
F
Robert
Vincent
V
Riedinger
Jean-Marc
JM
Stoltz
Jean-François
JF
Gambert
Philippe
P
Todd-Pokropek
Andrew
A
Lizard
Gérard
G
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
D005456:Fluorescent Dyes; D007653:Ketocholesterols; D010078:Oxazines; C003001:7-ketocholesterol; C044808:nile red