Single molecule spectroscopic characterization of GFP-MUT2 mutant for two-photon microscopy applications.
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.
Cannone
Fabio
F
Dipartimento di Fisica, Università di Milano Bicocca, Milano, Italy.
Caccia
Michele
M
Bologna
Sara
S
Diaspro
Alberto
A
Chirico
Giuseppe
G
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
D049452:Green Fluorescent Proteins; D058428:Silica Gel; D012822:Silicon Dioxide