title: Real-time phosphate sensing in living cells using fluorescence lifetime imaging microscopy (FLIM).
abstract: Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.
authors.affiliation: Department of Physical Chemistry, University of Granada.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Paredes
authors.forename: Maria D
authors.initials: MD
authors.lastname: Giron
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Ruedas-Rama
authors.forename: Angel
authors.initials: A
authors.lastname: Orte
authors.forename: Luis
authors.initials: L
authors.lastname: Crovetto
authors.forename: Eva M
authors.initials: EM
authors.lastname: Talavera
authors.forename: Rafael
authors.initials: R
authors.lastname: Salto
authors.forename: Jose M
authors.initials: JM
authors.lastname: Alvarez-Pez
mesh_terms: D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D010710:Phosphates / Q000737:chemistry*; D011522:Protons; D014966:Xanthenes / Q000737:chemistry; D044004:Xanthones / Q000737:chemistry
chemical_list: C543496:9-(1-(2-methyl-4-methoxyphenyl))-6-hydroxy-3H-xanthen-3-one; D005456:Fluorescent Dyes; D007477:Ions; D010710:Phosphates; D011522:Protons; D014966:Xanthenes; D044004:Xanthones