Dual Color Microscopic Imagery of Cells Expressing the Green
Fluorescent Protein and a Red-shifted Variant
Yang T.T., Kain S.R., Kitts P., Kondepudi A., Yang M.M., Youvan D.C.
Cell Biology Group, CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has
become a versatile reporter for monitoring gene expression and protein localization in a
variety of cells and organisms. GFP emits bright green light (lambda max = 510 nm) when
excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm).
The chromophore in GFP is intrinsic to the primary structure of the protein, and
fluorescence from GFP does not require additional gene products, substrates or other
factors. GFP fluorescence is stable, species-independent and can be monitored
noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie
et al., Science 263 (1994) 802-805; Stearns, Curr. Biol. 5 (1995) 262-264]. The protein
appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc.
Natl. Acad. Sci. USA 91 (1994) 12501-12504] in a process involving cyclization of a Tyr66
aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151-154], a combinatorial
mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A.
victoria GFP, yielding a number of different mutants with red-shifted fluorescence
excitation spectra. One of these, RSGFP4, retains the characteristic green emission
spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm). The
fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP
from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27
(1978) 389-396]. In the present study, we demonstrate by fluorescence microscopy that
selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each
fluorescent signal, and provides the means to image these signals independently in a mixed
population of bacteria or mammalian cells.