Individual green fluorescent proteins (GFP) studied by near-field optical microscopy

Abstract

Since the cloning and subsequent expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria research interest for this protein has increased dramatically. The exclusiveness of GFP as a biological marker derives from the finding that no cofactors are required for the protein’s fluorescence [1]. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be monitored by sensitive fluorescence methods without the need to add external fluorescent dyes. Other advantages of the protein include the possibility of performing genetic manipulation to alter its fluorescent properties, resulting in mutants that exhibit excitation and emission spectra different from those of the wild type (wt)-GFP and in some cases with higher quantum yield [2, 3]. Furthermore, it has been elucidated that the chromophore in the GFP is completely protected from bulk solvent [4]. The high quantum yield of fluorescence observed in ensemble measurements has been attributed to this presumably rigid encapsulation, while the low photodissociation rate observed for GFP has been attributed to the inability of O₂ to quench the excited state [4].