Abstract
Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.
Highlights
Lipid rafts due to their ability to distinguish Lo from liquid disordered phase (Ld) domains by changing fluorescence color, intensity, and lifetime
We have demonstrated an application of solvatochromic push-pull pyrene fluorophore, PA, to imaging of lipid membranes
Two-photon absorption cross section of PA is sufficiently high for two-photon excitation fluorescence microscopy, which reveals that PA can distinguish separated domains of ordered and disordered phases in giant unilamellar vesicles (GUVs)
Summary
Lipid rafts due to their ability to distinguish Lo from Ld domains by changing fluorescence color, intensity, and lifetime. PA (Fig. 1) shows the most attractive features such as high brightness (absorption coefficient is 25000 M−1 cm−1 in ethanol and fluorescence quantum yields, QY = 0.93 and 0.77 in hexane and ethanol, respectively), absorption band in the visible region (~420 nm), and strong solvatochromism (red shift from 480 nm to 598 nm on solvent change from hexane to ethanol)[35]. These photophysical properties of PA in organic solvents are superior to those of the commonly used Laurdan and Prodan derivatives, so that PA appears as a promising alternative membrane probe for lipid domains. PA revealed a clear change in the lipid order of cell membranes which decreased from the plasma membrane towards the nucleus
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