Terbium chelate membrane label for time-resolved, total internal reflection fluorescence microscopy of substrate-adherent cells

A chelating agent conjugated to a lipid was synthesized by reacting the dianhydride form of diethylenetriaminepentaacetic acid sequentially with 4-aminosalicylate and dioleoylphosphatidyl-ethanolamine. The product, DOPE-YAS-Tb, exhibits photophysical properties characteristic of a chelated Tb³⁺ ion bound to an organic triplet donor: an excitation maximum at 310 nm, narrow emission bands at 490, 545, 590, and 625 nm, and a lifetime of 1.57 ms. The suitability of DOPE-YAS-Tb as a membrane-staining agent for morphological studies of cultured cells using total internal reflection fluorescence microscopy (TIRFM) was investigated. Swiss albino mouse 3T3 cells were cultured on silica and polystyrene substrates. Time-resolved detection was employed to reject short-lived background emission (autoemission from the cells and/or the polymer substrate), which allowed the long-lived Tb³⁺ emission to be selectively imaged. The results show that time-resolved TIRFM of cells stained with DOPE-YAS-Tb is an effective method of quantitatively examining the cell morphology in situations where background due to autoemission from cells and/or the substrate material is problematic.