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Conference: Bucharest University Faculty of Physics 2011 Meeting

Section: Biophysics; Medical Physics

Title:

Fluidity Changes in Plasma Membranes of Cells Investigated by Laurdan and Two-Photon Excitation Fluorescence Lifetime Imaging Microscopy

Authors:

M. Bacalum(123), N. Smisdom(2), B. De Clercq(2), A. Popescu(3) , M.Radu(1) and M. Ameloot(2)

Affiliation:

1 Horia Hulubei National Institute of Research and Development in Physics and Nuclear Engineering (IFIN-HH), 30 Reactorului, Magurele-Ilfov, Romania

2 Biomedical Research Institute, Hasselt University, Agoralaan Bldg. C, B-3590 Diepenbeek, Belgium

3 Department of Electricity and Biophysics, Faculty of Physics, University of Bucharest, 405 Atomistilor, Magurele-Ilfov, Romania

E-mail

bmihaela@nipne.ro

Keywords:

Laurdan, FLIM,

Abstract:

Introduction: Due to its heterogeneous composition plasma membranes exhibit a variety of small (below optical diffraction limit) molecular domains, characterized by a specific lipid composition and spatial arrangement. In general two types of lipid domains are considered: i) a disordered lipid phase characterized by a low concentration of cholesterol and ii) an ordered lipid phase with a high cholesterol concentration. The fluorescent probe Laurdan was used to explore the membrane organization because of its characteristics: (i) a phase-dependent shift of almost 50 nm of the emission spectra maximum from ~440 nm in ordered lipid phases to ~500 nm in fluid phases, (ii) a phase dependent fluorescence lifetime (for ordered lifetimes are around 4-6 ns and for fluid phase the lifetimes are 2-3 ns [1], (iii) an equal partition between the ordered and disordered lipid phases, and (iv) a negligible fluorescence in water [2]. Materials and Methods: Laurdan was loaded into HEK-293 (Human Embryonic Kidney) and measurements were performed at 21oC and 37oC. Cholesterol depletion from the cells membranes was obtained by exposure to 10 mM methyl-β-cyclodextrin (MβCD) for 90 min. Two-photon excitation fluorescence microscopy was used. The probe was excited at 780 nm, and fluorescence intensity images and fluorescence lifetime images (FLIM) of the cross-section of the cells were collected simultaneously in two different channels, with emission in the range of 405–455 nm and 475–565 nm respectively [3]. The images were processed to obtain for each pixel the generalized polarization (GP) values [4] and the fluorescence lifetimes. Only the pixels from the membrane were selected by making a mask from the membrane. Results: The results show that both GP values and the fluorescence lifetimes are decreasing by the increase of temperature and/or the decrease of cholesterol concentration in the membrane. Thus GP values can be used to detect fluidity changes that appear in plasma membranes. The values decreased from 0.4 (21oC) to 0.35 (37oC) for normal cells and from 0.28 (21oC) to 0.23 (37oC) for cells treated with MβCD. In the case of fluorescence lifetimes significant changes were observed only after the cholesterol depletion of the membrane. References: [1] Yu et al., Biophys. J., 70: 626-636 (1996) [2] Bagatolli L. Biochim. Biophys. Acta 1758 (2006) 1541 [3] Schneckenburger H., et al., Photochem Photobiol Sci 3 (2004) 817 [4] Gaus K., et al., Molecular Membrane Biology, 23:41-48 (2006)