Multiphoton laser-scanning microscopy and spatial analysis of dehydroergosterol distributions on plasma membrane of living cells.
Multiphoton laser-scanning microscopy (MPLSM) imaging in combination with advanced image analysis techniques provides unique opportunities to visualize the arrangement of cholesterol in the plasma membrane (PM) of living cells. MPLSM makes possible the use of a naturally occurring sterol, dehydroergosterol (DHE), for observing sterol-enriched areas of the PM. Pure DHE has properties similar to cholesterol as observed in model and cellular membranes but with a conjugated double-bond system that fluoresces at ultraviolet wavelengths. MPLSM enables the excitation of DHE at infrared wavelengths that many laser-scanning microscopy systems are able to transmit effectively and that are less harmful to the cell. Thus, with the incorporation of DHE into living cells and the advent of MPLSM, real-time images of the cellular distribution of DHE can be obtained. In juxtaposition, notably the application of newly advanced techniques in image analysis, aids not only the identification and segmentation of sterol-rich regions of the PM of cells, but also the elucidation of the statistical nature of the observed patterns. In studies involving murine L-cell (Larpt-+K-) fibroblasts, DHE is shown to exhibit strong cluster patterns within the PM.
McIntosh
Avery L
AL
Dept. of Physiology and Pharmacology, Texas A & M University, College Station 77843, USA.
Atshaves
Barbara P
BP
Huang
Huan
H
Gallegos
Adalberto M
AM
Kier
Ann B
AB
Schroeder
Friedhelm
F
Xu
Hai
H
Zhang
Weimin
W
Wang
Suojin
S
Liu
Jyh-Charn
JC
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis; D007089:Image Enhancement; D007739:L Cells; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D010078:Oxazines / Q000737:chemistry; D017785:Photons; D053835:Unilamellar Liposomes / Q000378:metabolism
D005456:Fluorescent Dyes; D010078:Oxazines; D053835:Unilamellar Liposomes; C010905:dehydroergosterol; C044808:nile red; D004875:Ergosterol