Re: 532 nm excitation oid dyes
Howard Shapiro (hms@shapirolab.com)
Thu, 30 May 1996 19:26:44 -0400 (EDT)
532 nm is a really good wavelength for excitation of phycoerythrin, which
means any of your organisms which contain it will exhibit strong
autofluorescence, which may extend out as far as 700 nm depending on the
cells' content of phycocyanin, allophycocyanin, and chlorophyll.
For immunofluorescence and other labeling, PE and the PE-tandem conjugate
immunofluorescent labels obviously also excite very well at 532 nm, as do
TRITC and Cy3. You can get a little out of Texas Red with 532 nm
excitation, maybe not enough for sensitive immunofluorescence, but
definitely enough if you use it or its noncovalently binding relative SR101
as a total protein stain. PE-Cy5.5 (emission around 695) and PE-Cy7
conjugates (emission around 750) may be particularly interesting here.
Among nucleic acid stains, we have:
7-AAD (emission around 650), which has lousy quantum efficiency and
generally gives large CV's because of its sensitivity to chromatin
conformation, but which is DNA-specific;
ethidium and propidium (you can excite them with a 568 krypton laser, too),
which bind to DNA and RNA, as do:
LDS751, also known as styryl-8 (emission around 670),
PO-PRO-3 and POPO-3 (emission around 570), and
pyronin Y (good for RNA staining with Hoechst dyes or methyl green or
possibly 7-AAD to block DNA binding) (emission around 570),
and perhaps a few other cyanine, styryl, and tricyclic dyes, none of which
seems to be markedly better than what's listed above.
Fluorogenic enzyme substrates based on resorufin excite, though not that
well, at 532 nm.
Membrane potential dyes: Loew's rhodamine esters, DiICn(3) (cyanine),
DiTBACn(3) (oxonol), safranin.
pH Indicators: I think some of the SNARF or SNAFL dyes work here; ask
Molecular Probes.
I don't know about green-excited calcium probes.
Among membrane tracking dyes, "DiI" and PKH26 both excite at 532 nm (and
emit around 570.
For multiparameter work, you'd ideally like the weak labels to emit at the
shorter wavelengths, which probably isn't as easy to do with 532 nm
excitation as with 488 nm excitation. However, for mammalian cells, where
the autofluorescence excitation drops off precipitously above 510 nm, 532
excitation is really useful for high-sensitivity immunofluorescence
measurements, down in the range of 100 molecules.
-Howard
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