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 dioleoylphosphatidylethanolamine. 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.
Phimphivong
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Department of Chemistry, University of Arizona, Tucson 85721, USA.
Saavedra
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D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002614:Chelating Agents / Q000138:chemical synthesis*; D004396:Coloring Agents / Q000138:chemical synthesis; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D004369:Pentetic Acid / Q000138:chemical synthesis / Q000031:analogs & derivatives; D010714:Phosphatidylethanolamines / Q000138:chemical synthesis / Q000737:chemistry; D013053:Spectrophotometry; D013725:Terbium / Q000737:chemistry
D002614:Chelating Agents; D004396:Coloring Agents; C112702:DOPE YAS; D010714:Phosphatidylethanolamines; D013725:Terbium; D004369:Pentetic Acid