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, 90 (11), L80-2

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

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

Dylan M Owen et al. Biophys J.

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.

Figures

FIGURE 1
FIGURE 1
Fluorescence lifetime histograms for di-4-ANEPPDHQ in vesicles composed of DOPC and PSM/Chol, 7:3. (Inset) Structure of the probe.
FIGURE 2
FIGURE 2
(a) FLIM map and (b) red/green channel spectral merge of live HEK293 cells stained with di-4-ANEPPDHQ and imaged at 20°C (scale bar is 10 μm).
FIGURE 3
FIGURE 3
(Top) ROI lifetime histograms for di-4-ANEPPDHQ-stained HEK293 cells. (Bottom) The ROI FLIM maps used to produce the histograms (a) at 20°C, (b) 37°C, and (c) after 15-min incubation, with 7 mM MβCD at 37°C (scale bar is 10 μm).

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