One initial problem with the use of GFP was the excitation and emission spectra of the wild type protein for fluorescence microscopy. Wild type GFP has two excitation peaks, a major one at 395 nm (in the long UV range) and a smaller one at 475 nm (blue) and its emission peak at 509 nm (green). For wild type GFP, it has been found that exciting the protein at 395 nm causes fairly rapid quenching of the fluorescence. Also most investigators have used FITC filtersets to observe GFP staining. To alleviate this problem, several mutants of the GFP gene were constructed which have increased fluorescence, but perhaps more important, the major excitation peak has been red-shifted to 490 nm with the emission staying at 509 nm. This is better for use of FITC filtersets as this mutant GFP has the same excitation range as FITC. Furthermore, the main laserline used for FITC excitation is from the argon laser at 488nm. There is no good commonly used laserline near 395 nm in most confocals. One of the mutant GFPs which had a 5-6 times greater amount of fluorescence has a serine to threonine substitution at position 65 in the protein (S65T: See Heim, et al., Nature, 373: 663-664 (1995). Since then, other mutations have given further improvements in the brightness of the emission (See Clontech's website).
For general fluorescence microscopy purposes, investigators have been using normal FITC filtersets for viewing GFP. These are inadequate for both in that they are: Ex 485/20, Dichroic FT 510 , Em 520-560. (To review, for 485/20: 485 is the middle of the peak, /20 refers to the width of the band, 10 nm either side; FT 510 blocks all light below 510 nm and allows anything above that to pass).
In recent years, several different color forms of GFP have been produced. These are in order from shortest to longest emission spectra: blue, cyan, green, and yellow FP or BFP, CFP, GFP and YFP. One can now actually make double labeled specimens expressing fluorescently labeled proteins.
One manufacturer of fltersets for fluorescence microscopy, Chroma Technology has been specializing in making single and dual label filtersets for the various forms of GFP. They have produced a very useful brochure showing the spectral characteristics of these filtersets. The brochure is available for ordering or dowloading as a PDF file from their website Chroma Technology.
GFP and GFP color variant vectors for gene cloning and construction are available from Clontech.
At this time, it has been our experience that the pEGFP vectors from Clontech, when transfected into mammalian cells, produces a very bright GFP which works with the 488 nm laser line in the confocal microscope.
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