Single molecule spectroscopic characterization of GFP-MUT2 mutant for two-photon microscopy applications

Fabio Cannone, Michele Caccia, Sara Bologna, Alberto Diaspro, Giuseppe Chirico

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)186-193
Number of pages8
JournalMicroscopy Research and Technique
Volume65
Issue number4-5
DOIs
Publication statusPublished - Nov 2004

Keywords

  • Fluorescence
  • GFP
  • Photobleaching
  • Two-photon microscopy

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Anatomy
  • Instrumentation

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