Fluorescence Lifetime Imaging Provides Enhanced Contrast when Imaging the Phase-Sensitive Dye di-4-ANEPPDHQ in Model Membranes and Live Cells

Abstract

We apply fluorescence lifetime imaging to the membrane phase-sensing dye di-4-ANEPPDHQ in model membranes and live cells. We show that the 1700 ps lifetime shift between liquid-disordered and liquid-ordered phases offers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by observation of the temperature dependence of membrane order and by cholesterol depletion using methyl-beta-cyclodextrin.

Palmitoylation of MPP1 (Membrane-palmitoylated Protein 1)/p55 Is Crucial for Lateral Membrane Organization in Erythroid Cells

“…Fluorescence Lifetime Measurement by Time-correlated Single Photon Counting (TCSPC) FLIM-RBC were labeled with 7.5 M di-4-ANEPPDHQ (di-4) (Invitrogen) according to the protocol published by others (38,39). Fluorescence lifetime imaging microscopy (FLIM) was performed at 23°C with an LSM 510 microscope (Carl Zeiss) equipped with a FLIM dedicated system (PicoQuant).…”

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

“…These values are in agreement with those observed from the PM of Arabidopsis protoplasts, where GP ranged from -0.3 to 0.2 immediately after enzymatic digestion of the cell wall, up to a value of 0.6 15 h later (Blachutzik et al, 2012). A similar broad histogram distribution was previously described using fluorescence lifetime imaging in live epithelial cells labeled with di-4-ANEPPDHQ (Owen et al, 2006), as well as laurdan-labeled K562 cells (Balogh et al, 2011). Combined fluorescence anisotropy and polarization-resolved fluorescence imaging revealed a similar histogram distribution in di-8-ANEPPQ orientational order measured in COS7 cells (Kress et al, 2011).…”

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties

“…Both di-4-ANEPPDHQ ( 31,32 ) and Laurdan ( 33,34 ) have proved to be extremely sensitive to changes in membrane order through changes in their fl uorescence emission spectra or lifetimes. Both molecules sense the reorientation of solvent dipoles, whose strength is directly dependent on membrane fl uidity, this being related to water penetration within membranes ( 31,33 ).…”

“…3D-F ). Decreases in membrane ordering are expected to induce a signifi cant decrease in fl uorescence lifetime of di-4-ANEPPDHQ in living cells ( 32 ), and FLIM measurements were carried out in HEK293T cells before and after a 30 min exposure to bile acids ( Fig. 3D ).…”