Green (and other) Fluorescent Proteins
THE CENTRAL MICROSCOPY FACILITY
The University of Massachusetts
Amherst, Massachusetts USA 01003
"Green Fluorescent Protein" (GFP) has become a very useful tool in cellular and molecular biology research. The DNA sequence for GFP can be inserted into the DNA of an organism in such a way that GFP is produced whenever the gene of interest is expressed. The ability to detect very small amounts of a compound by fluorescence microscopy gives the researcher the ability to have a very sensitive assay for gene expression and the localization of gene products.
Since the first use of GFP (wild-type) that was derived from jellyfish, several variants of GFP have been introduced that have differences in spectral characteristics to make them more suitable for specific purposes. Some of those specific purposes are: to improve the sensitivity of our ability to detect GFP by tuning the excitation and emission spectrum of a GFP derivative to better match the excitation of common lasers or arc lamp sources; to provide a higher quantum yield; to provide better separation between the excitation and emission spectra; and to provide fluorophores that are spectrally separated for multiple-label studies. An excellent source of information for spectra and detailed characteristics of a selection of available GFP derivatives can be found at the Clontech company's GFP webpages (no endorsement implied!).
In the following discussion, the designations mentioned refer to the product descriptions given in the Clontech literature (purely for a common reference point) and would equally well apply to products of similar spectral characteristics from any other source. Please refer to the spectra from the Clontech online Living Colors Pamphlet. Download (free!) the latest Adobe Acrobat Reader for most operating systems if you need it to view Acrobat PDF files.
Enhanced Blue Fluorescent Protein (EBFP) is also very well suited for the Nikon UV-1A filter set we use for DAPI visualization on the fluorescence microscopes. This could be used for a double label system with EGFP using our current filter sets (UV-1A and B-1A). Unfortunately this is not a useful system with our confocal system (KrAr laser) and it should be noted that the EBFP bleaches more rapidly than the others.
ECFP ("Enhanced Cyan Fluorescent Protein") can be used with our fluorescence microscopes. The filter set for this dye uses the 405 nm Hg line for excitation and uses an DF510/40 emission filter for an excellent match to the emission band.
With our current filter sets, the "enhanced GFP" (EGFP) is a very favorable probe because it is efficiently excited by the 488nm line of the BioRad Krypton-Argon laser for those using the Bio-Rad MRC-600 Laser Scanning Confocal Microscope (LSCM), and the emission is efficiently processed and detected . It also works very well on the standard epi-fluorescence microscope using a 100 watt Hg arc lamp and the standard "FITC" filter block; even though there is no Hg line matching the peak excitation, the continuum in the 470-490 nm passband is adequate and the emission is efficiently passed to the detector. Chlorophyll autofluorescence will sneak past the barrier (510LP) with this filter set: we have ordered a bandpass filter to eliminate this problem.
In summary, for use with standard ("wide-field") epi-fluorescence microscopy the user can currently choose from EBFP, ECFP, or EGFP with the filter sets we have on hand. This is not to say that we can spectrally isolate these probes in a multilabelling scenario. Specific narrow band filter sets would have to be obtained to separate these probes. Other filter sets could be assembled for use with EYFP (yellow emission) or to get a higher discrimination between the EBFP, ECPF, and EGFP. For the confocal microscope we are limited to the use of EGFP (most efficiently) due to the laser line at 488 nm, but it does look nice!
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