title: Single molecule spectroscopic characterization of GFP-MUT2 mutant for two-photon microscopy applications.
abstract: Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.
authors.affiliation: Dipartimento di Fisica, Università di Milano Bicocca, Milano, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Cannone
authors.forename: Michele
authors.initials: M
authors.lastname: Caccia
authors.forename: Sara
authors.initials: S
authors.lastname: Bologna
authors.forename: Alberto
authors.initials: A
authors.lastname: Diaspro
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Chirico
mesh_terms: D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009154:Mutation; D038761:Photobleaching; D017785:Photons; D058428:Silica Gel; D012822:Silicon Dioxide; D013057:Spectrum Analysis
chemical_list: D049452:Green Fluorescent Proteins; D058428:Silica Gel; D012822:Silicon Dioxide