Pyrene excimer mapping in cultured fibroblasts by ratio imaging and time-resolved microscopy

Abstract

The kinetics of pyrene eximer formation provide a measure of lateral diffusibility in bilayer membranes. Swiss 3T3 fibroblasts were labeled with pyrene, pyrenedecanoic acid (PDA) and 1,3-bis(1 pyrene) propane (BPP) by incubation in the presence of Pluronic F127. Single-cell emission spectra obtained by epifluorescence microscopy (excitation 350 nm) with photodiode array detection showed monomer (380-420 nm) and eximer (475 nm) peaks. The eximer-to-monomer fluorescence ratio (E/M) increased with increasing temperature and loading time. Time-resolved microscopy studies of fibroblasts labeled with PDA for 15 min gave monomer and eximer lifetimes of 101 and 78 ns, respectively, with a monomer-to-eximer conversion rate of 0.02 ns-1. E/M ratio images were obtained with a microchannel plate intensifier and CCD camera at 350-nm excitation and 405 +/- 5 nm (monomer) and greater than 470-nm (eximer) emission wavelengths. E/M ratios of PDA showed spatial variation across the cell with highest ratios at the peripheral plasma membrane. These results establish the methodology to label cells with pyrene eximer-forming probes and to image eximer distributions in membranes of intact cultured cells. Eximer-to-monomer fluorescence ratios are sensitive to maneuvers that alter the membrane physical state and should be of utility in examining the cellular regulation of membrane fluidity.