Hi David,
You should talk with Paul Millman of Chroma Technology (800-824-7662)
because he may already have your ideal filter for your GFP variant (I'm
pretty sure he has the "Piston" filter set). Two papers you may want to
review:
M.J. Zylka, B.J. Schnapp (1996) Optimized filter sets and viewing
conditions for the S65T mutant of GFP in living cells. BioTechniques
21:220-226. [ex: 470-490 nm, dm 505 nm, em 510-532 nm, our MetaMorph
software, cooled CCD]
K.D. Niswender, S.M. Blackman, L. Rhode, M.A. Magnuson, D.W. Piston (1995)
Quantitative imaging of green fluorescent protein in cultured cells:
comparison of microscopic techniques, use in fusion proteins and detection
limits. J. Microscopy 180:109-116. ("Piston" filter set).
If you work on mammalian fibroblasts or similar "generic" tissue culture
cells, wavelengths longer than UV are likely to have equal effect. Short
wavelengths are more likely to excite autofluorescence (NADH and
flavoproteins) than long wavelengths. Your 490 nm filter should be fine.
You are correct in thinking that a wide pass emission filter is good
(asuming no autofluorescence and no other fluorochromes in use).
One factor that I have not seen much comment on is matching the GFP
variant spectrum and emission filter to the detector quantum efficiency.
Most scientific grade detectors have a maximum quantum efficiency that does
not extend over the whole visible range. For example, among digital CCD
cameras, the "standard" Kodak KAF-1400 CCD has a quantum efficiency of
about 15% at 450 nm, 20% at 500 nm and 40% from 550-600 nm. This suggests
that for a given signal-to-noise ratio, that BFP (450 nm emission) images
would be acquired much less efficiently than the most red-shifted FP's
(S65T and longer emission maxima). There is nothing intrinsically "bad"
about the KAF-1400 Q.E. at 450 nm, it is simply a fact that needs to be
taken into account in choosing your fluorochrome (by the way, video CCD's
have much lower QE at all wavelengths. Standard intensifier's also have a
low QE in ther blue. For caomparison, there are back-illuminated CCD's with
quantum efficienies of better than 70% QE throughout the visible light
range). KAF-1400 owners would be better off with Clontech's GFPuv (520 nm
emission) than with BFP, i.e. for sequential dual wavelength experiments
with GFP-S65T.
For spectral shift issues see:
M. Chattoraj, B.A. King, G.U. Bublitz, S.G. Boxer (1996) Ultra-fast excited
state dynamics in green fluorescent protein: multiple states and proton
transfer. Proc. Natl. Acad. Sci. USA 93: 8362-8367.
G.J. Palm, A. Zdanov, G.A. Gaitanaris, R. Stauber, G.N. Pavlakis, A.
Wlodawer (1997) The structural basis for spectral variation in green
fluorescent protein. Nature Biotechnology 4: 361-365.
K. Brejc, T.K.Sixma, P.A. Kitts, S.R. Kain, R.Y. Tsien, M. Ormo, S.J.
Remington (1997) Structural basis for dual excitation and
photoisomerization of the Aequorea victoria green fluorescent protein. Proc
Natl Acad Sci U S A 94: 2306-2311.
F. Yang, L.G. Moss, G.N. Phillips Jr. (1996) The molecular structure of
green fluorescent protein. Nature Biotechnology 14:1246-1251.
F. Yang, L.G. Moss, G.N. Phillips Jr. (1997) The three-dimensional
structure of green fluorescent protein in Bioluminescence and
Chemiluminescence: Molecular Reporting with Photons, J.W. Hastings, L.J.
Kricka, P.E. Stanley, eds. John Wiley & Sons, Chichester. pages 375-382.
I found the Palm et al paper to be the most readable of these.
My understanding is that the chromophore is well protected by the
surrounding barrel and that amino acid changes like S65T and Y66H lock the
chromophore into one or the other excitation (360-410 or 470-500 nm) and
emission states (450 vs 470-520 nm).
Good luck,
Sincerely,
geo
>I'm trying to design a fluorescence filter set to view GFP fusion proteins
>in live cells. I want to illuminate a narrow wavelength range centered on
>the 488 488nm excitation peak of S65T. I'm considering an excitation
>bandpass of 490 +/- 10nm, 505nm dichroic and 535 +/- 25nm emission
>filters. My intention is to reduce phototoxicity of live cells by reducing
>illumination at shorter wavelengths. But I could get in trouble with such
>narrow excitation range if excitation or emission spectra are changed upon
>fusion of GFP to other proteins, or when the fusion is in particular
>intracellular environments. Does anyone know whether GFP spectra change
>much? Other suggestions? Thanks!
>>--David Roof
>>*****************************************************************
>David Roof, Ph.D. Phone: 215-573-3636
>Department of Physiology FAX: 215-573-5851
>D202 Richards email: Roof at mail.med.upenn.edu>University of Pennsylvania
>3700 Hamilton Walk
>Philadelphia, PA 19104-6085
>*****************************************************************
>>
George McNamara, Ph.D.
Applications Scientist
Universal Imaging Corporation
502 Brandywine Parkway
West Chester, PA 19380 USA
voice: 610-344-9410 ext 224
fax: 610-344-9515
internet: http://www.image1.com
GFP: http://www.image1.com/products/metagfp/green.html
