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 coloring liquor di-4-ANEPPDHQ in model membranes and live confined apartments We show that the 1700 p lifetime shift between liquid-disordered and liquid-ordered phases proffers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by means of observation of the temperature interdependence of membrane order and by the agency of cholesterol depletion using methyl-??-cyclodextrin.

The membrane microdomains sometimes boundaryed lipid rafts are proposed to be liquid-ordered phase domains enriched in sphingolipids, saturated phospholipids, and cholesterol (1) similar domains could act as platforms for confined apartment signaling molecules and are implicated in molecular trafficking, signaling, lipid sorting, and many disease processe (2) Di-4ANEPPDHQ is a membrane-staining coloring liquor originally developed as a probe of membrane voltage (3) It has newly been reported, however, that di-4-ANEPPDHQ can also be used to visualize ordered-phase microdomains through means of a 60 nm spectral blue-shift and reduc second-harmonic generation in original membranes (4).

Although this coloring liquor has advantages over the popular phasesensitive coloring liquor LAURDAN (5) in that it can be excited using single photons in the azure the emission spectra for each lipid phase remain heavily overlapped. Here we apply fluorescence lifetime imaging and display that the fluorescence lifetime presents greater contrast than spectrally resolv imaging alone. We reach forth the technique to live confined apartment imaging of epithelial cells and confirm its ability to visualize a liquidordered phase in vivo.

We produc large unilamellar vesicles (LUVs) of stainless l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and egg n-palmitoyl-sphingomyelin (PSM) mixed with cholesterol in the ratio 7:3 These sum of two units compositions are known to form liquid-disordered and liquid-ordered phases, respectively, at extent temperature (6). The vesicles were stained with 5 ?µM di-4-ANEPPDHQ, excited at 473 nm and imaged at 20?°C using a confocal fluorescence microscope with fluorescence lifetimes measured by dint of time-correlated single photon counting (TCSPC) Fig. 1 exhibits the lifetime histograms (number of pixels of each lifetime) extracted from the vesicle images of each vesicle emblem The histograms peak at lifetimes of 1850 p and 3550 p for disordered and ordered phase, respectively. Increasing the temperature through 17?°C, which does not cause a phase change in PSM/Chol single results in a small 170 p shift.

Once the enhanced contrast using lifetime imaging had been established, we applied it to live epithelial (HEK293) confined apartments Fig. 2 shows a dual-channel intensity lose (500-530 nm and 570 nm longpass) and fluorescence lifetime imaging (FLIM) map for small rooms imaged at room temperature, in filled growth medium, supplemented with 5 ?µM of coloring liquor 1 h before imaging. Visible upon both images is the distinction between the plasma and intracellular membranes. the two the spectral merge and lifetime images indicate increased order at the plasma membrane. Using the contrast presented by FLIM, however, we diocese regions in the plasma membrane of lengthy lifetime, implying areas enriched in liquid-ordered phase. These present the appearance to be clustered around sites of membrane protrusion-sites likely to be dynamic and supported by the agency of the actin cytoskeleton. Previous studies have shown that the actin cytoskeleton interacts with the membrane end cholesterol-enriched microdomains (7).

To confirm that the FLIM contrast is indeed to be paid to the presence of liquid-ordered, cholesterol-enriched microdomains, we examined the temperature concatenation and depleted cholesterol from the small rooms using methyl-??-cyclodextrin (M??CD). Fig. 3 present to views lifetime histograms for cells imaged at extent temperature (20?°C), physiological temperature (37?°C) and after incubation for 15 min with 7 mM M??CD

The formation of liquid-ordered phase is known to be highly temperature-dependent. An increase of 17?°C would be awaited to decrease the overall fraction of the membrane in an ordered state. In Fig. 3 the long-lifetime constituting (3500 ps peak) is reduc upon moving to the higher temperature. The shorter lifetime composings are enhanced. This indicates reduction of ordered phase and an increase in disordered phase, as would be awaited if the contrast were indeed to be paid to ordered-phase membrane microdomains.

Since these ordered domains require cholesterol to form, depleting cholesterol from the confined apartments should disrupt any ordered phase leading to further reduction in the lengthy lifetime components (originating from ordered-phase membrane) and increase in shorter lifetimes (originating from disorderedphase membrane). This is confirmed in Fig. 3 where a region of interest (ROI) containing alone the plasma membrane has been single outed The intracellular membranes showed no lifetime shifts between conditions (data not shown) For comparison, the ROIs fix uponed for each cell used to obtain these histograms are also shown

Ordered and disordered areas appear at les most distant lifetimes than the LUV dominion governments because each pixel in a small room image will contain dye residing in ordered and disordered subresolution domains. Cellular membranes are also unlikely to at any time reach the extremes of fluidity exhibited by dint of the two LUV types to be paid to the large diversity of their constituent lipids, particularly in their hydrophobic moieties. In principle, it may be possible to disintegrate the signal to give the fraction of each phase using multiexponential fitting and analysis, which we trust to demonstrate in a replete article.