Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy.
Curr Biol
; 9(5): 269-72, 1999 Mar 11.
Article
em En
| MEDLINE
| ID: mdl-10074454
ABSTRACT
The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.
Buscar no Google
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Luminescentes
/
Microscopia de Fluorescência
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
1999
Tipo de documento:
Article